Antibacterial 4,5-substituted aminoglycoside analogs having multiple substituents

ABSTRACT

The present invention is directed to analogs of aminoglycoside compounds as well as their preparation and use as prophylactic or therapeutics against microbial infection.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/987,842, filed Jan. 10, 2011, now pending, which is a continuation ofU.S. patent application Ser. No. 12/130,048, filed May 30, 2008, nowissued as U.S. Pat. No. 7,893,039, which is a continuation ofInternational PCT Patent Application No. PCT/US2006/046122, which wasfiled on Dec. 1, 2006, now abandoned, which claims the benefit under 35U.S.C. §119(e) of U.S. Provisional Patent Application No. 60/742,051filed Dec. 2, 2005. These applications are incorporated herein byreference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to novel aminoglycoside compounds andsynthetic methods for their preparation and use as therapeutic orprophylactic agents.

2. Description of the Related Art

A particular interest in modern drug discovery is the development ofnovel low molecular weight orally-bioavailable drugs that work bybinding to RNA. RNA, which serves as a messenger between DNA andproteins, was thought to be an entirely flexible molecule withoutsignificant structural complexity. Recent studies have revealed asurprising intricacy in RNA structure. RNA has a structural complexityrivaling proteins, rather than simple motifs like DNA. Genome sequencingreveals both the sequences of the proteins and the mRNAs that encodethem. Since proteins are synthesized using an RNA template, suchproteins can be inhibited by preventing their production in the firstplace by interfering with the translation of the mRNA. Since bothproteins and the RNAs are potential drug targeting sites, the number oftargets revealed from genome sequencing efforts is effectively doubled.These observations unlock a new world of opportunities for thepharmaceutical industry to target RNA with small molecules.

Classical drug discovery has focused on proteins as targets forintervention. Proteins can be extremely difficult to isolate and purifyin the appropriate form for use in assays for drug screening. Manyproteins require post-translational modifications that occur only inspecific cell types under specific conditions. Proteins fold intoglobular domains with hydrophobic cores and hydrophilic and chargedgroups on the surface. Multiple subunits frequently foim complexes,which may be required for a valid drug screen. Membrane proteins usuallyneed to be embedded in a membrane to retain their proper shape. Thesmallest practical unit of a protein that can be used in drug screeningis a globular domain. The notion of removing a single alpha helix orturn of a beta sheet and using it in a drug screen is not practical,since only the intact protein may have the appropriate 3-dimensionalshape for drug binding. Preparation of biologically active proteins forscreening is a major limitation in classical high throughput screening.Quite often the limiting reagent in high throughput screening efforts isa biologically active form of a protein which can also be quiteexpensive.

For screening to discover compounds that bind RNA targets, the classicapproaches used for proteins can be superceded with new approaches. AllRNAs are essentially equivalent in their solubility, ease of synthesisor use in assays. The physical properties of RNAs are independent of theprotein they encode. They may be readily prepared in large quantitythrough either chemical or enzymatic synthesis and are not extensivelymodified in vivo. With RNA, the smallest practical unit for drug bindingis the functional subdomain. A functional subdomain in RNA is a fragmentthat, when removed from the larger RNA and studied in isolation, retainsits biologically relevant shape and protein or RNA-binding properties.The size and composition of RNA functional subdomains make themaccessible by enzymatic or chemical synthesis. The structural biologycommunity has developed significant experience in identification offunctional RNA subdomains in order to facilitate structural studies bytechniques such as NMR spectroscopy. For example, small analogs of thedecoding region of 16S rRNA (the A-site) have been identified ascontaining only the essential region, and have been shown to bindantibiotics in the same fashion as the intact ribosome.

The binding sites on RNA are hydrophilic and relatively open as comparedto proteins. The potential for small molecule recognition based on shapeis enhanced by the deformability of RNA. The binding of molecules tospecific RNA targets can be determined by global conformation and thedistribution of charged, aromatic, and hydrogen bonding groups off of arelatively rigid scaffold. Properly placed positive charges are believedto be important, since long-range electrostatic interactions can be usedto steer molecules into a binding pocket with the proper orientation. Instructures where nucleobases are exposed, stacking interactions witharomatic functional groups may contribute to the binding interaction.The major groove of RNA provides many sites for specific hydrogenbonding with a ligand. These include the aromatic N7 nitrogen atoms ofadenosine and guanosine, the O4 and O6 oxygen atoms of uridine andguanosine, and the amines of adenosine and cytidine. The rich structuraland sequence diversity of RNA suggests to us that ligands can be createdwith high affinity and specificity for their target.

Although our understanding of RNA structure and folding, as well as themodes in which RNA is recognized by other ligands, is far from beingcomprehensive, significant progress has been made in the last decade(Chow, C. S.; Bogdan, F. M., Chem. Rev., 1997, 97, 1489, Wallis, M. G.;Schroeder, R., Prog. Biophys. Molec. Biol. 1997, 67, 141). Despite thecentral role RNA plays in the replication of bacteria, drugs that targetthese pivotal RNA sites of these pathogens are scarce. The increasingproblem of bacterial resistance to antibiotics makes the search fornovel RNA binders of crucial importance.

Certain small molecules can bind and block essential functions of RNA.Examples of such molecules include the aminoglycoside antibiotics anddrugs such as erythromycin which binds to bacterial rRNA and releasespeptidyl-tRNA and mRNA. Aminoglycoside antibiotics have long been knownto bind RNA. They exert their antibacterial effects by binding tospecific target sites in the bacterial ribosome. For the structurallyrelated antibiotics neamine, ribostamycin, neomycin B, and paromomycin,the binding site has been localized to the A-site of the prokaryotic 16Sribosomal decoding region RNA (Moazed, D.; Noller, H. F., Nature, 1987,327, 389). Binding of aminoglycosides to this RNA target interferes withthe fidelity of mRNA translation and results in miscoding andtruncation, leading ultimately to bacterial cell death (Alper, P. B.;Hendrix, M.; Sears, P.; Wong, C., J. Am. Chem. Soc., 1998, 120, 1965).

There is a need in the art for new chemical entities that work againstbacteria with broad-spectrum activity. Perhaps the biggest challenge indiscovering RNA-binding antibacterial drugs is identifying vitalstructures common to bacteria that can be disabled by small moleculedrug binding. A challenge in targeting RNA with small molecules is todevelop a chemical strategy which recognizes specific shapes of RNA.There are three sets of data that provide hints on how to do this:natural protein interactions with RNA, natural product antibiotics thatbind RNA, and man-made RNAs (aptamers) that bind proteins and othermolecules. Each data set, however, provides different insights to theproblem.

Several classes of drugs obtained from natural sources have been shownto work by binding to RNA or RNA/protein complexes. These include threedifferent structural classes of antibiotics: thiostreptone, theaminoglycoside family and the macrolide family of antibiotics. Theseexamples provide powerful clues to how small molecules and targets mightbe selected. Nature has selected RNA targets in the ribosome, one of themost ancient and conserved targets in bacteria. Since antibacterialdrugs are desired to be potent and have broad-spectrum activity theseancient processes fundamental to all bacterial life represent attractivetargets. The closer we get to ancient conserved functions the morelikely we are to find broadly conserved RNA shapes. It is important toalso consider the shape of the equivalent structure in humans, sincebacteria were unlikely to have considered the therapeutic index of theirRNAs while evolving them.

A large number of natural antibiotics exist, these include theaminoglycosides, kirromycin, neomycin, paromomycin, thiostrepton, andmany others. They are very potent, bactericidal compounds that bind RNAof the small ribosomal subunit. The bactericidal action is mediated bybinding to the bacterial RNA in a fashion that leads to misreading ofthe genetic code. Misreading of the code during translation of integralmembrane proteins is thought to produce abnormal proteins thatcompromise the barrier properties of the bacterial membrane.

Antibiotics are chemical substances produced by various species ofmicroorganisms (bacteria, fungi, actinomycetes) that suppress the growthof other microorganisms and may eventually destroy them. However, commonusage often extends the term antibiotics to include syntheticantibacterial agents, such as the sulfonamides, and quinolines, that arenot products of microbes. The number of antibiotics that have beenidentified now extends into the hundreds, and many of these have beendeveloped to the stage where they are of value in the therapy ofinfectious diseases. Antibiotics differ markedly in physical, chemical,and pharmacological properties, antibacterial spectra, and mechanisms ofaction. In recent years, knowledge of molecular mechanisms of bacterial,fungal, and viral replication has greatly facilitated rationaldevelopment of compounds that can interfere with the life cycles ofthese microorganisms.

At least 30% of all hospitalized patients now receive one or morecourses of therapy with antibiotics, and millions of potentially fatalinfections have been cured. At the same time, these pharmaceuticalagents have become among the most misused of those available to thepracticing physician. One result of widespread use of antimicrobialagents has been the emergence of antibiotic-resistant pathogens, whichin turn has created an ever-increasing need for new drugs. Many of theseagents have also contributed significantly to the rising costs ofmedical care.

When the antimicrobial activity of a new agent is first tested, apattern of sensitivity and resistance is usually defined. Unfortunately,this spectrum of activity can subsequently change to a remarkabledegree, because microorganisms have evolved the array of ingeniousalterations discussed above that allow them to survive in the presenceof antibiotics. The mechanism of drug resistance varies frommicroorganism to microorganism and from drug to drug.

The development of resistance to antibiotics usually involves a stablegenetic change, heritable from generation to generation. Any of themechanisms that result in alteration of bacterial genetic compositioncan operate. While mutation is frequently the cause, resistance toantimicrobial agents may be acquired through transfer of geneticmaterial from one bacterium to another by transduction, transformationor conjugation.

For the foregoing reasons, there is a need for new chemical entitiesthat possess antimicrobial activity. Further, in order to accelerate thedrug discovery process, new methods for synthesizing aminoglycosideantibiotics are needed to provide an array of compounds that arepotentially new drugs for the treatment microbial infections.

BRIEF SUMMARY OF THE INVENTION

The present invention provides compounds having the following formula I:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof,

wherein:

Q₁ is azido, —OH, a protected hydroxyl, —NR₂R₃ or a nitrogen containingheterocycle radical which can include one or more additional heteroatomsselected from N, O and S wherein the heterocycle is covalently linkedthrough said nitrogen atom;

Q₂ Q₂ is —NR₂R₄;

each Q₃ and Q₄ is —OR₇;

Q₅ is H, halogen, cyano, azido, —OR₈, —NR₂R₃, a protected amino group ora nitrogen containing heterocyclic radical which can include one or moreadditional heteroatoms selected from N, O and S wherein the heterocyclicradical is covalently linked through said nitrogen atom;

each R₁ is, independently, H or a hydroxyl protecting group;

each R₂ is, independently, H, an amino protecting group, C₁-C₁₂ alkyl orsubstituted C₁-C₁₂ alkyl;

each R₃ is, independently, H, an amino protecting group, cyano, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl or—(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁;

R₄ is H, an amino protecting group, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl or a group having the following formula III:

each R₆ is, independently, H or an amino protecting group;

each R₇ is, independently, H, a hydroxyl protecting group or—(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁;

R₈ is H, a hydroxyl protecting group, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl or —(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁;

L₁ is S, O or NJ₁;

L₂ is CH or N;

n is an integer from 1 to 8;

m is 0 or 1;

nn is 0 or an integer from 1 to 8;

mm is 1 or 2;

E₁ is H, hydroxyl, halogen, cyano, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heteroaryl, substituted heteroaryl, aheterocyclic radical, a substituted heterocyclic radical or asubstituted or unsubstituted mono or poly cyclic structure that can beunsaturated, partially saturated or fully saturated and can include oneor more heteroatoms selected from O, N and S;

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl,—C(═O)—X, a heterocyclic radical or a substituted heterocyclic radical;

each X is, independently, H, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl;

each Z₁ and Z₂ is, independently, H, hydroxyl or a protected hydroxyl;and

Z₃ is —OR₈ or a group having the following formula IV:

and

wherein at least two of Q₁, Q₂, Q₃, Q₄ and Q₅ are other than hydroxyl,protected hydroxyl, amino or a protected amino group and when Q₂ is—N(H)C(═O)C(H)(OH)CH₂—CH₂NH₂ then Q₁ is other than —N(H)CH₃ or—N(H)CH₂CH₃ and Q₅ is other than —N(H)C(═NH)NH₂ or —N(H)CH═NH₂.

In one aspect of the present invention, the compounds of formula I aresubstituted such that:

Q₁ is azido or —NR₁₀R₁₁; and

Q₂ is —NR₁₀R₁₂,

wherein:

R₁₀ is H, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl;

R₁₁ is cyano, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynylor —(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁; and

R₁₂ is C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl or a group having formulaIII.

In another aspect of the present invention, the compounds of formula Iare substituted such that:

Q₁ is azido or —NR₁₀R₁₁; and

Q₃ is —OR₁₃,

wherein:

R₁₀ is H, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl;

R₁₁ is cyano, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynylor —(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁; and

R₁₃ is —(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁.

In another aspect of the present invention, the compounds of formula Iare substituted such that:

Q₁ is azido or —NR₁₀R₁₁; and

Q₄ is —OR₁₃,

wherein:

R₁₀ is H, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl;

R₁₁ is cyano, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynylor —(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁; and

R₁₃ is —(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁.

In another aspect of the present invention, the compounds of formula Iare substituted such that:

Q₁ is azido or —NR₁₀R¹¹; and

Q₅ is halogen, cyano, azido, —OR₁₄ or —NR₁₀R₁₁,

wherein:

R₁₀ is H, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl;

R₁₁ is cyano, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynylor —(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁; and

R₁₄ is C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynylor —(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁.

In another aspect of the present invention, the compounds of formula Iare substituted such that:

Q₂ is —NR₁₀R₁₂, and

Q₃ is —OR₁₃,

wherein:

R₁₀ is H, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl;

R₁₂ is C₁-C₁₂ alkyl, substituted C₁-C₁₂ alky or a group having formulaIII; and

R₁₃ is —(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁.

In another aspect of the present invention, the compounds of formula Iare substituted such that:

Q₂ is —NR₁₀R₁₂; and

Q₄ is —OR₁₃,

wherein:

R₁₀ is H, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl;

R₁₂ C₁-C₁₂ alkyl, substituted C₁-C₁₂ alky or a group having formula III;and

R₁₃ is —(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁.

In another aspect of the present invention, the compounds of formula Iare substituted such that:

Q₂ is —NR₁₀R₁₂; and

Q₅ is halogen, cyano, azido, —OR₁₄ or —NR₁₀R₁₁;

wherein:

R₁₀ is H, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl;

R₁₁ is cyano, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynylor —(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)E₁;

R₁₂ is C₁-C₁₂ alkyl, substituted C₁-C₁₂ alky or a group having formulaIII; and

R₁₄ is C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynylor —(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁.

In another aspect of the present invention, the compounds of formula Iare substituted such that Q₃ and Q₄ are each —OR₁₃, wherein each R₁₃ is,independently, —(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁.

In another aspect of the present invention, the compounds of formula Iare substituted such that:

Q₃ is —OR₁₃; and

Q₅ is halogen, cyano, azido, —OR₁₄ or —NR₁₀R₁₁;

wherein:

R₁₃ is —(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁;

R₁₀ is H, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl;

R₁₁ is cyano, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynylor —(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁; and

R₁₄ is C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynylor —(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁.

In another aspect of the present invention, the compounds of formula Iare substituted such that:

Q₄ is —OR₁₃; and

Q₅ is halogen, cyano, azido, —OR₁₄ or —NR₁₀R₁₁;

wherein:

R₁₀ is H, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl;

R₁₁ is cyano, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynylor —(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁;

R₁₃ is —(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁; and

R₁₄ is C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynylor —(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁.

In another aspect of the present invention, the compounds of formula Iare substituted such that Z₁ and Z₂ are both H.

In another aspect of the present invention, the compounds of formula Iare substituted such that Z₁ and Z₂ are both hydroxyl or protectedhydroxy.

In another aspect of the present invention, the compounds of formula Iare substituted such that one of Z₁ and Z₂ is H.

In another aspect of the present invention, the compounds of formula Iare substituted such that Q₂ is an optionally protected group having theformula —N(H)C(O)C(H)(OH)(CH₂)₂NH₂.

In another aspect of the present invention, the compounds of formula Iare substituted such that Z₃ is —OR₈.

In another aspect of the present invention, the compounds of formula Iare substituted such that Z₃ is a group having formula IV.

In another aspect of the present invention, the compounds of formula Iare substituted such that:

Q₂ is:

and

Q₄ is —O—(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁.

In another aspect of the present invention, the compounds of formula Iare substituted such that:

Q₁ is —OH, a protected hydroxyl or —NR₂R₃;

each Q₃ and Q₅ is, independently, —OH or a protected hydroxyl; at leastone of Z₁ and Z₂ is H;

Z₃ is a group having formula IV; and

each R₂ is, independently, H or an amino protecting group.

In another aspect of the present invention, the compounds of formula Iare substituted such that:

Q₁ is amino or protected amino;

L₂ is CH;

L₁ is —NJ₁; and

E₁ is C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heteroaryl, substitutedheteroaryl or a substituted or unsubstituted mono or poly cyclicstructure that can be unsaturated, partially saturated or fullysaturated and can include one or more heteroatoms selected from O, N andS.

In another aspect of the present invention, the compounds of formula Iare substituted such that:

n is an integer from 1 to 3;

m is 1; and

nn is an integer from 1 to 3.

In another aspect of the present invention, the compounds of formula Iare substituted such that E₁ is C₅-C₂₀ aryl or substituted C₅-C₂₀ aryl.

In another aspect of the present invention, the compounds of formula Iare substituted such that E₁ is phenyl.

In another aspect of the present invention, the compounds of formula Iare substituted such that: n is 2; and nn is 2.

In another aspect of the present invention, the compounds of formula Iare substituted such that each of Z₁ and Z₂ is H.

In another aspect of the present invention, the compounds of formula Iare substituted such that one of Z₁ and Z₂ is H and the other of Z₁ andZ₂ is hydroxy.

In another aspect of the present invention, the compounds of formula Iare substituted such that Q₂ has the configuration:

and

-   -   * indicates a chiral carbon having (S)-configuration.

In another aspect of the present invention, the compounds of formula Iare substituted such that:

Q₂ is:

Q₄ is —O—(CH₂)₂—N(H)—(CH₂)₂—C₆H₅;

Q₁ is —OH, a protected hydroxyl, amino or protected amino;

Q₃ and Q₅ are each —OH;

Z₃ is a group having formula IV; and

at least one of Z₁ and Z₂ is H.

In another aspect of the present invention, the compounds of formula Iare substituted such that Q₂ and Q₄ are each, independently, a groupother than hydroxyl, protected hydroxyl, amino or protected amino.

In another aspect of the present invention, the compounds of formula Iare substituted such that Q₁ is —OH, protected hydroxyl or —NR₂R₃.

In another aspect of the present invention, the compounds of formula Iare substituted such that Z₃ is a group having formula IV.

In another aspect of the present invention, the compounds of formula Iare substituted such that:

each of said substituted groups, is, independently, mono or polysubstituted with optionally protected substituent groups independentlyselected from C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl,C₅-C₂₀ aryl or substituted C₅-C₂₀ aryl, heterocyclic radical,substituted heterocyclic radical, heteroaryl, substituted heteroaryl,C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclic radical, halogen,—OJ₃, —SJ₃, —N₃, —COOH, —C(═O)—X, —CN, —S(═O)₂—X, —S(═O)—X,—C(═O)—NJ₁J₂, —N(H)C(═O)-J₁, —N(J₁)—(CH₂)_(nm)—OJ₃ and—N(J₁)-(CH₂)_(nm)—NJ₁J₂ and a substituted or unsubstituted mono or polycyclic structure that can be unsaturated, partially saturated or fullysaturated and can include one or more heteroatoms selected from O, N andS;

each J₃ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl,C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substitutedC₂-C₁₂ alkynyl, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl or ahydroxyl protecting group; and

nm is an integer from 1 to 20.

In another aspect of the present invention, the compounds of formula Ihave the configuration:

In another aspect of the present invention, the compounds of formula Ihave the configuration:

The present invention also provides methods of using a compound of theinvention in therapy. In particular, the present invention provides amethod of treating a bacterial infection in a mammal comprisingadministering to the mammal an effective amount of a compound of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, in one aspect of the present invention, aminoglycosidecompounds are provided having the following formula I:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof,

wherein:

Q₁ is azido, —OH, a protected hydroxyl, —NR₂R₃ or a nitrogen containingheterocycle radical which can include one or more additional heteroatomsselected from N, O and S wherein the heterocycle is covalently linkedthrough said nitrogen atom;

Q₂ is —NR₂R₄;

each Q₃ and Q₄ is —OR₇;

Q₅ is H, halogen, cyano, azido, —OR₈, —NR₂R₃, a protected amino group ora nitrogen containing heterocyclic radical which can include one or moreadditional heteroatoms selected from N, O and S wherein the heterocyclicradical is covalently linked through said nitrogen atom;

each R₁ is, independently, H or a hydroxyl protecting group;

each R₂ is, independently, H, an amino protecting group, C₁-C₁₂ alkyl orsubstituted C₁-C₁₂ alkyl;

each R₃ is, independently, H, an amino protecting group, cyano, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl or—(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁;

R₄ is H, an amino protecting group, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl or a group having the following formula

each R₆ is, independently, H or an amino protecting group;

each R₇ is, independently, H, a hydroxyl protecting group or—(CH₂)_(n)-(L₁)_(m)(CH₂)_(nn)-E₁;

R₈ is H, a hydroxyl protecting group, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl or or —(CH₂)_(n)-(L)_(n)-(CH₂)_(nn)-E₁;

L₁ is S, O or NJ₁;

L₂ is CH or N;

n is an integer from 1 to 8;

m is 0 or 1;

nn is 0 or an integer from 1 to 8;

mm is 1 or 2;

E₁ is H, hydroxyl, halogen, cyano, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heteroaryl, substituted heteroaryl, aheterocyclic radical, a substituted heterocyclic radical or asubstituted or unsubstituted mono or poly cyclic structure that can beunsaturated, partially saturated or fully saturated and can include oneor more heteroatoms selected from O, N and S;

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl,—C(═O)—X, a heterocyclic radical or a substituted heterocyclic radical;

each X is, independently, H, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl;

each Z₁ and Z₂ is, independently, H, hydroxyl or a protected hydroxyl;and

Z₃ is —OR₈ or a group having the following formula IV:

and

wherein at least two of Q₁, Q₂, Q₃, Q₄ and Q₅ are other than hydroxyl,protected hydroxyl, amino or a protected amino group and when Q₂ is—N(H)C(═O)C(H)(OH)CH₂—CH₂NH₂ then Q₁ is other than —N(H)CH₃ or—N(H)CH₂CH₃ and Q₅ is other than —N(H)C(═NH)NH₂ or —N(H)CH═NH₂.

In other aspects of the present invention, wherein Z₃ is —OR₈ or Z₃ is agroup having the formula IV, aminoglycoside compounds are providedhaving the following formulas V and VI, respectively:

In more specific embodiments of the foregoing, aminoglycoside compoundsare provided having the following formulas VII and VIII, respectively:

wherein:

each Z_(a) and Z_(b) is, independently, H, —OH or a protected hydroxyl;

each R_(a) is, independently, H, a hydroxyl protecting group or an aminoprotecting group;

at least two of Q_(a), Q_(b), Q_(c), Q_(d) and Q_(e) are, independently,an optionally linked chemical functional group; and

each of the remaining Q_(a), Q_(b), Q_(c), Q_(d) and Q_(e) are,independently, hydroxyl, amino, a protected hydroxyl, a protected aminoor an optionally linked chemical functional group.

Aminoglycoside compounds of the present invention are prepared accordingto established organic synthetic methods. In a particular generalmethod, paromomycin is selectively protected such that one of the 1, 2″,5″, 6 or 6′ positions can be selectively functionalized followed bydeprotection of one of the remaining protected positions for furtherfunctionalization. Following the orthogonal protection schemes providedin the examples below aminoglycoside compounds are prepared having atleast two of the 1, 2″, 5″, 6 or 6′ positions selectivelyfunctionalized.

In a preferred embodiment the compounds of the present invention areprepared from paromomycin sulfate salt (commercially available fromvarious sources including Sigma-Aldrich Co., et al.). The reactivegroups are orthogonally protected as illustrated in the examples belowto enable selective functionalization of at least two of the 1, 2″, 5″,6 or 6′ positions. The methods disclosed herein are amenable to a widevariety of chemical reactions to prepare a large number of paromomycinanalogs. The present invention therefor provides a variety ofsubstituted paromomycin analogs that are useful as therapeutic and/orprophylactic agents as well as processes and inteimmediates for makingthem.

In some preferred embodiments each of the 3′ and 4′ substituents (eitherZ₁ and Z₂ or Z_(a) and Z_(b)) are hydroxyl groups as found inparomomycin. In other embodiments one or both of the 3′ and 4′substituents are hydrogen.

The term “chemical functional group” as used herein, refers to one ormore groups that are directly attached or linked to a site in acompound. Such groups can enhance the properties of the parent compoundto provide for example enhanced activity against one or more selectedtargets. A representative list of chemical functional groups includes,but is not limited to, H, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aminoalkyl, substitutedaminoalkyl, carbocyclic alkyl, substituted carbocyclic alkyl, alkenylcarbocyclic, substituted alkenyl carbocyclic, alkynyl carbocyclic,substituted alkynyl carbocyclic, aryl, substituted aryl, aralkyl,substituted aralkyl, —O-aralkyl, —S-aralkyl, —NH-aralkyl, heteroaryl,substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, aheterocycle containing one or more heteroatoms selected from N, O and S,a substituted heterocycle, alicyclyl, substituted alicyclyl, asubstituted or unsubstituted mono or poly cyclic structure that can beunsaturated, partially saturated or fully saturated and can include oneor more heteroatoms selected from O, N and S, wherein said mono or polycyclic structure is bonded directly or through said substituent group,hydroxyl, alkoxy, thiol, thioalkyl, halogen, an ether having 2 to 10carbon atoms and 1 to 4 oxygen or sulfur atoms, a metal coordinationgroup, a conjugate group, trifluoromethyl, trifluoromethoxy, —OJ_(a),—C(═O)J_(c), ═O, —C(═O)OJ_(c), —NJ_(a)J_(b), ═NJ_(a),—N(J_(a))C(═O)J_(c), —N(J_(a))C(═O)NJ_(a)J_(b),—N(J_(a))C(S)NJ_(a)J_(b), —N(J_(a))S(O)₂J_(a),—N(J_(a))C(═NJ_(a))NJ_(a)J_(b), —N(J_(a))(CH₂)_(nmn)—OJ_(b),—N(J_(a))(CH₂)_(nmn)NJ_(a)J_(b), —C(═O)NJ_(a)J_(b), —OC(═O)NJ_(a)J_(b),—C(═NJ_(a))NJ_(a)J_(b), —C(═NJ_(a))J_(a),—C(═O)—(CH₂)₂—CH(NJ_(a)J_(b))—C(═O)OJ_(a), —CN, —NO₂, —N₃, —NHNH₂,—ONH₂, —S(O)₂NJ_(a)J_(b), —S(O)₂J_(a), S, —SJ_(a), silyl, an amino acidside chain, a carbohydrate, a drug, or a group capable of hydrogenbonding where nmn is from 1 to about 20.

Wherein each J_(a) and J_(b) is, independently, H, C₁-C₂₀ alkyl,substituted C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, substituted C₂-C₂₀ alkenyl,C₂-C₂₀ alkynyl, substituted C₂-C₂₀ alkynyl, C₅-C₂₀ aryl, substitutedC₅-C₂₀ aryl, a heterocyclic radical, a substituted heterocyclic radical,heteroaryl, substituted heteroaryl, C₁-C₁₂ aminoalkyl, substitutedC₁-C₁₂ aminoalkyl, —C(O)J_(c), a protecting group, an optionally linkedconjugate group or an optionally linked chemical functional group.

Wherein each J_(c) is, independently, H, hydroxyl, C₁-C₂₀ alkyl,substituted C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, substituted C₂-C₂₀ alkenyl,C₂-C₂₀ alkynyl, substituted C₂-C₂₀ alkynyl, C₅-C₂₀ aryl, substitutedC₅-C₂₀ aryl, a heterocyclic radical, a substituted heterocyclic radical,heteroaryl, substituted heteroaryl, C₁-C₁₂ aminoalkyl, substitutedC₁-C₁₂ aminoalkyl, a protecting group, an optionally linked conjugategroup or an optionally linked chemical functional group.

The terms “substituent” and “substituent group,” as used herein, aremeant to include groups that are typically added to other groups orparent compounds to enhance desired properties or give desired effects.Substituent groups can be protected or unprotected and can be added toone available site or to many available sites in a parent compound.Substituent groups may also be further substituted with othersubstituent groups and may be attached directly or via a linking groupsuch as an alkyl or hydrocarbyl group to a parent compound. Suchsubstituent groups include without limitation, halogen, hydroxyl, alkyl,alkenyl, alkynyl, acyl (—C(O)R_(aa)), carboxyl (—C(O)O—R_(aa)),aliphatic groups, alicyclic groups, alkoxy, substituted oxo (—O—R_(aa)),aryl, aralkyl, heterocyclic, heteroaryl, heteroarylalkyl, amino(—NR_(bb)R_(cc)), imino (═NR_(bb)), amido (—C(O)NR_(bb)R_(cc) or—N(R_(bb))C(O)R_(aa)), azido (—N₃), nitro (—NO₂), cyano (—CN), carbamido(—OC(O)NR_(bb)R_(cc) or —N(R_(bb))C(O)OR_(aa)), ureido(—N(R_(bb))C(O)NR_(bb)R_(cc)), thioureido (—N(R_(bb))C(S)NR_(bb)R_(cc)),guanidinyl (—N(R_(bb))C(—NR_(bb))NR_(bb)R_(cc)), amidinyl(—C(═NR_(bb))NR_(bb)R_(cc) or —N(R_(bb))C(NR_(bb))R_(aa)), thiol(—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)),sulfonamidyl (—S(O)₂NR_(bb)R_(cc) or —N(R_(bb))S(O)₂R_(bb)) andconjugate groups. Wherein each R_(aa), R_(bb) and R_(cc) is H, anoptionally linked chemical functional group or a further substituentgroup, with a preferred list including, without limitation, H, alkyl,alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl,alicyclic, heterocyclic and heteroarylalkyl groups.

Linking groups such as those known in the art are amenable to thepresent invention. Linking groups or bifunctional linking moieties areuseful for attachment of chemical functional groups, conjugate groups,reporter groups and other groups to selective sites in a parentcompound. In general a bifunctional linking moiety comprises ahydrocarbyl moiety having two functional groups. One of the functionalgroups is selected to bind to a parent molecule or compound of interestand the other is selected to bind essentially any selected group such aschemical functional group or a conjugate group. In some embodiments, thelinker comprises a chain structure or an oligomer of repeating unitssuch as ethylene glyol or amino acid units. Examples of functionalgroups that are routinely used in a bifunctional linking moietiesinclude, but are not limited to, electrophiles for reacting withnucleophilic groups and nucleophiles for reacting with electrophilicgroups. In some embodiments, bifunctional linking moieties includeamino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double ortriple bonds), and the like. Some nonlimiting examples of bifunctionallinking moieties include 8-amino-3,6-dioxaoctanoic acid (ADO),succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and6-aminohexanoic acid (AHEX or AHA). Other linking groups include, butare not limited to, substituted C₁-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl or substituted or unsubstituted C₂-C₁₀alkynyl, wherein a nonlimiting list of preferred substituent groupsincludes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

The term “hydrocarbyl” includes groups comprising C, O and H. Includedare straight, branched and cyclic groups having any degree ofsaturation. Such hydrocarbyl groups can include one or more heteroatomsselected from N, O and S and can be further mono or poly substitutedwith one or more substituent groups.

The term “alkyl,” as used herein, refers to a saturated straight orbranched hydrocarbon radical containing up to twenty four carbon atoms.Examples of alkyl groups include, but are not limited to, methyl, ethyl,propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.Alkyl groups typically include from 1 to about 24 carbon atoms (C₁-C₂₄alkyl), more typically from 1 to about 12 carbon atoms (C₁-C₁₂ alkyl)with from 1 to about 6 carbon atoms (C₁-C₆ alkyl) being more preferred.The teen “lower alkyl” as used herein includes from 1 to about 6 carbonatoms. Alkyl groups as used herein may optionally include one or morefurther substitutent groups.

The term “alkenyl,” as used herein, refers to a straight or branchedhydrocarbon chain radical containing from two up to twenty four carbonatoms and having at least one carbon-carbon double bond. Examples ofalkenyl groups include, but are not limited to, ethenyl, propenyl,butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and thelike. Alkenyl groups typically include from 2 to about 24 carbon atoms(C₂-C₂₄ alkenyl), more typically from 2 to about 12 carbon atoms (C₂-C₁₂alkenyl) with from 2 to about 6 carbon atoms (C₂-C₆ alkenyl) being morepreferred. Alkenyl groups as used herein may optionally include one ormore further substitutent groups.

The term “alkynyl,” as used herein, refers to a straight or branchedhydrocarbon radical containing from two up to twenty four carbon atomsand having at least one carbon-carbon triple bond. Examples of alkynylgroups include, but are not limited to, ethynyl, 1-propynyl, 1-butynyl,and the like. Alkynyl groups typically include from 2 to about 24 carbonatoms (C₂-C₂₄ alkynyl), more typically from 2 to about 12 carbon atoms(C₂-C₁₇ alkynyl) with from 2 to about 6 carbon atoms (C₂-C₆ alkynyl)being more preferred. Alkynyl groups as used herein may optionallyinclude one or more further substitutent groups.

The term “aminoalkyl” as used herein, refers to an amino substitutedalkyl, alkenyl or alkynyl radical. This term is meant to include C₁-C₁₂alkyl groups having an amino substituent at any position and wherein thealkyl group attaches the aminoalkyl group to the parent molecule. Thealkyl, alkenyl, alkynyl or amino portions of the aminoalkyl group can befurther substituted with substituent groups.

The term “aliphatic,” as used herein, refers to a straight or branchedhydrocarbon radical containing up to twenty four carbon atoms whereinthe saturation between any two carbon atoms is a single, double ortriple bond. An aliphatic group preferably contains from 1 to about 24carbon atoms, more typically from 1 to about 12 carbon atoms with from 1to about 6 carbon atoms being more preferred. The straight or branchedchain of an aliphatic group may be interupted with one or moreheteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Suchaliphatic groups interupted by heteroatoms include without limitationpolyalkoxys, such as polyalkylene glycols, polyamines, and polyimines.Aliphatic groups as used herein may optionally include furthersubstitutent groups.

The term “alicyclic” or “alicyclyl” refers to a cyclic ring systemwherein the ring is aliphatic. The ring system can comprise one or morerings wherein at least one ring is aliphatic. Preferred alicyclicsinclude rings having from about 5 to about 9 carbon atoms in the ring.Alicyclic as used herein may optionally include further substitutentgroups.

The term “alkoxy,” as used herein, refers to a radical formed between analkyl, alkenyl or alkynyl group and an oxygen atom wherein the oxygenatom is used to attach the alkoxy group to a parent molecule. Examplesof alkoxy groups include, but are not limited to, methoxy, ethoxy,propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy,neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein mayoptionally include further substitutent groups.

The terms “halo” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

The terms “aryl” and “aromatic,” as used herein, refer to a mono- orpolycyclic carbocyclic ring system radicals having one or more aromaticrings. Examples of aryl groups include, but are not limited to, phenyl,naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferredaryl ring systems have from about 5 to about 20 carbon atoms in one ormore rings. Aryl groups as used herein may optionally include furthersubstitutent groups.

The terms “aralkyl” and “arylalkyl,” as used herein, refer to a radicalformed between an alkyl, alkenyl or alkynyl group and an aryl groupwherein the alkyl, alkenyl or alkynyl group is used to attach thearalkyl group to a parent molecule. Examples include, but are notlimited to, benzyl, phenethyl and the like. Aralkyl groups as usedherein may optionally include further substitutent groups attached tothe alkyl, alkenyl, alkynyl, aryl or both groups that foim the radicalgroup.

The term “heterocyclic,” “heterocyclic radical,” or “heterocycle” asused herein, refers to a radical mono-, or poly-cyclic ring system thatincludes at least one heteroatom and is unsaturated, partially saturatedor fully saturated, thereby including heteroaryl groups. Heterocyclic isalso meant to include fused ring systems wherein one or more of thefused rings contain at least one heteroatom and the other rings cancontain one or more heteroatoms or optionally contain no heteroatoms. Aheterocyclic group typically includes at least one atom selected fromsulfur, nitrogen or oxygen. Examples of heterocyclic groups include,[1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl,imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl,morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl,pyridazinonyl, tetrahydrofuryl and the like. Heterocyclic groups as usedherein may optionally include further substitutent groups.

The terms “heteroaryl,” and “heteroaromatic,” as used herein, refer to aradical comprising a mono- or poly-cyclic aromatic ring, ring system orfused ring system wherein at least one of the rings is aromatic andincludes one or more heteroatom. Heteroaryl is also meant to includefused ring systems including systems where one or more of the fusedrings contain no heteroatoms. Heteroaryl groups typically include onering atom selected from sulfur, nitrogen or oxygen. Examples ofheteroaryl groups include, but are not limited to, pyridinyl, pyrazinyl,pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl,isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl,isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and thelike. Heteroaryl radicals can be attached to a parent molecule directlyor through a linking moiety such as an aliphatic group or hetero atom.Heteroaryl groups as used herein may optionally include furthersubstitutent groups.

The term “heteroarylalkyl,” as used herein, refers to a heteroaryl groupas previously defined having an alkyl, alkenyl or alkynyl radical thatcan attach the heteroarylalkyl group to a parent molecule. Examplesinclude, but are not limited to, pyridinylmethyl, pyrimidinylethyl,napthyridinylpropyl and the like. Heteroarylalkyl groups as used hereinmay optionally include further substitutent groups.

The term “mono or poly cyclic structure” as used in the presentinvention includes all ring systems that are single or polycyclic havingrings that are fused or linked and is meant to be inclusive of singleand mixed ring systems individually selected from aliphatic, alicyclic,aromatic, aralkyl, heterocyclic, heteroaromatic, and heteroarylalkylgroups. Such mono and poly cyclic structures can contain rings that areuniform or have varying degrees of saturation including fully saturated,partially saturated or fully unsaturated. Each ring can comprise ringatoms selected from C, N, O and S to give rise to heterocyclic rings aswell as rings comprising only C ring atoms which can be present in amixed motif such as for example benzimidazole wherein one ring has onlycarbon ring atoms and the fused ring has two nitrogen atoms. The mono orpoly cyclic structures can be further substituted with substituentgroups such as for example phthalimide which has two ═O groups attachedto one of the rings. In another aspect, mono or poly cyclic structurescan be attached to a parent molecule directly through a ring atom,through a substituent group or a bifunctional linking moiety.

The term “acyl,” as used herein, refers to a radical formed by removalof a hydroxyl group from an organic acid and has the general formula—C(O)—X, where X is typically aliphatic, alicyclic or aromatic. Acylgroups as used herein may optionally include further substitutentgroups.

In one aspect of the present invention aminoglycoside compounds havingformula I, V and VI are are modified by covalent attachment of one ormore conjugate groups that modify one or more properties of thecompounds, including but not limited to pharmakodynamic,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and clearance. Conjugate groups are routinely used in thechemical arts with a preferred list including, without limitation,intercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, thioethers, polyethers, cholesterols, thiocholesterols, cholicacid moieties, folate, lipids, phospholipids, biotin, phenazine,phenanthridine, anthraquinone, adamantane, acridine, fluoresceins,rhodamines, coumarins and dyes. Reporter groups that are suitable asconjugate groups include any moiety that can be detected by, forexample, spectroscopic means. Examples of reporter groups include dyes,fluorophores, phosphors, radiolabels, and the like. In some embodiments,the reporter group is biotin, flourescein, rhodamine, coumarin, orrelated compounds. Reporter groups can also be attached to otherconjugate moieties. Conjugate moieties can be attached directly to acompound of the present invention or through a linker group orbifunctional linking moiety (linker or tether).

The term “protecting group,” as used herein, refers to a labile chemicalmoiety which is known in the art to protect reactive groups includingwithout limitation, hydroxyl, amino and thiol groups, against undesiredreactions during synthetic procedures. Protecting groups are typicallyused selectively and/or orthogonally to protect sites during reactionsat other reactive sites and can then be removed to leave the unprotectedgroup as is or available for further reactions. Protecting groups asknown in the art are described generally in Greene and Wuts, ProtectiveGroups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York(1999).

Groups can be selectively incorporated into aminoglycosides of theinvention as precursors. For example an amino group can be placed into acompound of the invention as an azido group that can be chemicallyconverted to the amino group at a desired point in the synthesis.Generally, groups are protected or present as a precursor that will beinert to reactions that modify other areas of the parent molecule forconversion into their final groups at an appropriate time. Furtherrepresentative protecting or precursor groups are discussed in Agrawal,et al., Protocols for Oligonucleotide Conjugates, Eds, Humana Press; NewJersey, 1994; Vol. 26 pp. 1-72.

Examples of hydroxyl protecting groups include, but are not limited to,t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl,p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl,diphenylmethyl, p-nitrobenzyl, triphenylmethyl, trimethylsilyl,triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl,triphenylsilyl, benzoylformate, acetate, chloroacetate,trichloroacetate, trifluoroacetate, pivaloate, benzoate,p-phenylbenzoate, 9-fluorenylmethyl carbonate, mesylate and tosylate.

Examples of amino protecting groups include, but are not limited to,carbamate-protecting groups, such as 2-trimethylsilylethoxycarbonyl(Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl(BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc),and benzyloxycarbonyl (Cbz); amide protecting groups, such as formyl,acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl;sulfonamide-protecting groups, such as 2-nitrobenzenesulfonyl; and imineand cyclic imide protecting groups, such as phthalimido anddithiasuccinoyl.

Examples of thiol protecting groups include, but are not limited to,triphenylmethyl (trityl), benzyl (Bn), and the like.

The synthesized compounds can be separated from reaction mixtures andfurther purified by methods including but not limited to columnchromatography, high pressure liquid chromatography andrecrystallization. Further methods of synthesizing the compounds of theformulae herein will be evident to those of ordinary skill in the art.Additionally, the various synthetic steps may be performed in analternate sequence or order to give the desired compounds. Syntheticchemistry transformations and protecting group methodologies (protectionand deprotection) useful in synthesizing the compounds described hereinare known in the art and include, for example, those such as describedin R. Larock, Comprehensive Organic Transformations, VCH Publishers(1989); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

The compounds described herein contain one or more asymmetric centersand thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)-, α or β, or as (D)- or (L)- such as foramino acids et al. The present invention is meant to include all suchpossible isomers, as well as their racemic and optically pure forms.Optical isomers may be prepared from their respective optically activeprecursors by the procedures described above, or by resolving theracemic mixtures. The resolution can be carried out in the presence of aresolving agent, by chromatography or by repeated crystallization or bysome combination of these techniques which are known to those skilled inthe art. Further details regarding resolutions can be found in Jacques,et al., Enantiomers, Racemates, and Resolutions (John Wiley & Sons,1981). When the compounds described herein contain olefinic doublebonds, other unsaturation, or other centers of geometric asymmetry, andunless specified otherwise, it is intended that the compounds includeboth E and Z geometric isomers or cis- and trans-isomers. Likewise, alltautomeric forms are also intended to be included. The configuration ofany carbon-carbon double bond appearing herein is selected forconvenience only and is not intended to designate a particularconfiguration unless the text so states; thus a carbon-carbon doublebond or carbon-heteroatom double bond depicted arbitrarily herein astrans may be cis, trans, or a mixture of the two in any proportion.

Susceptible organisms generally include those gram positive and gramnegative, aerobic and anaerobic organisms whose growth can be inhibitedby the compounds of the invention such as Staphylococcus, Lactobacillus,Streptococcus, Sarcina, Escherichia, Enterobacter, Klebsiella,Pseudomonas, Acinetobacter, Proteus, Campylobacter, Citrobacter,Nisseria, Baccillus, Bacteroides, Peptococcus, Clostridium, Salmonella,Shigella, Serratia, Haemophilus, Brucella and other organisms.

It has been found that the compounds of the present invention possessantibacterial activity against a wide spectrum of gram positive and gramnegative bacteria, as well as enterobacteria and anaerobes. Thecompounds, by reason of their in vitro activity, may be used in scrubsolutions for surface inhibition of bacterial growth, e.g., insterilization of glasswear or as an additive in fabric launderingcompositions.

Accordingly there is provided a method of treating bacterial infectionin a mammal comprising administering to the mammal, for example a human,an effective amount of a compound of the invention. By “effectiveamount” is meant an amount of compound which upon administration iscapable of reducing or preventing proliferation of the bacteria orreducing or preventing symptoms associated with the bacterial infection.The actual amount of compound administered and the route ofadministration will depend upon the particular disease or bacteria aswell as other factors such as the size, age, sex and ethnic origin ofthe individual being treated and is determined by routine analysis. Thecompounds of the invention may also be formulated into compositionstogether with pharmaceutically acceptable carriers for parenteralinjection, for oral administration in solid or liquid form, for rectaladministration, and the like. In methods of the invention, the compoundmay be administered orally (including buccal, sublingual, inhalation),nasally, rectally, vaginally, intravenously, intradermally,subcutaneously and topically. Compounds will be formulated intocompositions suitable for administration for example with suitablecarriers, diluents, thickeners, adjuvants, etc., as are routine in theformulation art. Compositions of the invention may also includeadditional active ingredients. Dosage forms include solutions, powders,tables, capsules, gel capsules, suppositories, topical ointments andcreams and aerosols for inhalation.

Formulations for non-parenteral administration may include sterileaqueous solutions which may also contain buffers, diluents and othersuitable additives. Pharmaceutically acceptable organic or inorganiccarrier substances suitable for non-parenteral administration which donot deleteriously react with compounds of the invention can be used.Suitable pharmaceutically acceptable carries include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike. The formulations can be sterilized and, if desired, mixed withauxiliary agents, e.g., lubricants, preservatives, stabilizers, wettingagents, emulsifiers, salts for influencing osmotic pressure, buffers,colorings flavorings and/or aromatic substances and the like which donot deleteriously react with compounds of the invention. Aqueoussuspensions may contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. Optionally, the suspension may also containstabilizers.

In a preferred embodiment, compounds of the invention are administeredvia oral delivery. Compositions for oral administration include powdersor granules, suspensions or solutions in water or non-aqueous media,capsules, sachets, troches, tablets or SECs (soft elastic capsules orcaplets). Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids, carrier substances of binders may be desirably added tosuch formulations. The use of such formulations has the effect ofdelivering the nucleic acid to the alimentary canal for exposure to themucosa thereof. Accordingly, the formulation can consist of materialeffective in protecting the compound from pH extremes of the stomach, orin releasing the compound over time, to optimize the delivery thereof toa particular mucosal site. Enteric coatings for acid-resistant tablets,capsules and caplets are known in the art and typically include acetatephthalate, propylene glycol and sorbitan monoleate.

Various methods for producing formulations for alimentary delivery arewell known in the art. See, generally, Nairn, Chapter 83; Block, Chapter87; Rudnic et. al., Chapter 89; and Longer et. al., Chapter 91 In:Remington's Pharmaceutical Sciences, 18^(th) Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990. The formulations of the invention canbe converted in a known manner into the customary fog mulations, such astablets, coated tablets, pills, granules, aerosols, syrups, emulsions,suspensions and solutions, using inert, non-toxic, pharmaceuticallysuitable excipients or solvents. The therapeutically active compoundshould in each case be present in a concentration of about 0.5% to about95% by weight of the total mixture, that is to say in amounts which aresufficient to achieve the desired dosage range. The formulations areprepared, for example, by extending the active compounds with solventsand/or excipients, if appropriate using emulsifying agents and/ordispersing agents, and, for example, in the case where water is used asthe diluent, organic solvents can be used as auxiliary solvents ifappropriate.

Compositions may be formulated in a conventional manner using additionalpharmaceutically acceptable carriers or excipients as appropriate. Thus,the composition may be prepared by conventional means with additionalcarriers or excipients such as binding agents (e.g., pregelatinisedmaize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose);filters (e.g., lactose, microcrystalline cellulose or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc or silica);disintegrates (e.g., starch or sodium starch glycolate); or wettingagents (e.g., sodium lauryl sulfate). Tablets may be coated by methodswill known in the art. The preparations may be also contain flavoring,coloring and/or sweetening agents as appropriate.

The pharmaceutical formulations, which may conveniently be presented inunit dosage form, may be prepared according to conventional techniqueswell known in the pharmaceutical industry. Such techniques include thestep of bringing into association the active ingredients with thepharmaceutical carrier(s) or excipient(s). In general the formulationsare prepared by uniformly and intimately bringing into association theactive ingredients with liquid carriers or finely divided soled carriersor both, and then, if necessary, shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tableseach containing predetermined amounts of the active ingredients; aspowders or granules; as solutions or suspensions in an aqueous liquid ora non-aqueous liquid; or as oil-in-water emulsions or water-in-oilliquid emulsions. A tablet may be made by compression or molding,optionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing in a suitable machine, the activeingredients in a free-flowing form such as a powder or granules,optionally mixed with a binder, lubricant, inert diluent, preservative,surface active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredients therein. Included withinthe scope of the present invention are the pharmaceutically acceptablesalts of the foregoing compounds. As used herein, the term“pharmaceutically acceptable salts” refers to non-toxic acid additionsalts and alkaline earth metal salts of the compounds of the invention.The salts can be prepared in situ during the final isolation andpurification of the compounds of the invention, or separately byreacting the free base or acid functions with a suitable organic acid orbase. Representative acid addition salts include the hydrochloride,hydrobromide, sulphate, bisulphate, acetate, oxalate, valerate, oleate,palmitate, stearate, laurate, borate, benzoate, lactate, phosphate,tosylate, mesylate, citrate, maleate, fumarate, succinate, tartrate,glucoheptonate, lactobionate, lauryl sulfate salts and the like.Representative alkali or alkaline earth metal salts include the sodium,calcium, potassium and magnesium salts.

Included within the scope of the present invention are prodrugs of theforegoing compounds. As used herein, the term “prodrug” refers to acompound that may be converted under physiological conditions or bysolvolysis to a biologically active compound of the present invention.Thus, the term “prodrug” refers to a metabolic precursor of a compoundof the present invention that is pharmaceutically acceptable. A prodrugmay be inactive when administered to a subject in need thereof, but isconverted in vivo to an active compound. Prodrugs are typically rapidlytransformed in vivo to yield the active compound, for example, byhydrolysis in blood. The prodrug compound often offers advantages ofsolubility, tissue compatibility or delayed release in a mammalianorganism (see, e.g., Bundgard, H., Design of Prodrugs (1985), pp. 7-9,21-24 (Elsevier, Amsterdam)). A discussion of prodrugs is also providedin Higuchi, T., et al., “Pro-drugs as Novel Delivery Systems,” A.C.S.Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design,Ed. Edward B. Roche, American Pharmaceutical Association and PergamonPress, 1987, both of which are incorporated in full by reference herein.

The term “prodrug” is also meant to include any covalently bondedcarriers, which release an active compound of the present invention invivo when such prodrug is administered to a mammalian subject. Prodrugsare generally prepared by modifying functional groups in a way such thatthe modification is cleaved, either by routine manipulation or in vivo,yielding the parent compound. Prodrugs include, for example, compoundsof the present invention wherein hydroxy, amine or sulfhydryl groups arebonded to any group that, when administered to a mammalian subject,cleaves to form the hydroxy, amine or sulfhydryl groups. Thus,representative examples of prodrugs include (but are not limited to)acetate, formate and benzoate derivatives of alcohol and aminefunctional groups of the compounds of the present invention. Further, inthe case of a carboxylic acid (—COOH), esters may be employed, such asmethyl esters, ethyl esters, and the like.

The invention disclosed herein is also meant to encompass the in vivometabolic products of the disclosed compounds. Such products may resultfrom, for example, the oxidation, reducation, hydrolysis, amidation,esterification, and the like of the administered compound, primarily dueto enzymatic processes. Accordingly, the invention includes compoundsproduced by a process comprising contacting a compound of this inventionwith a mammal for a period of time sufficient to yield a metabolicproduct thereof. Such products are typically are identified byadministering a radiolabelled compound of the invention in a detectabledose to an animal, such as rat, mouse, guinea pig, monkey, or to human,allowing sufficient time for metabolism to occur, and isolating itscoversion products from the urine, blood or other biological samples.

EXAMPLES Example 1 4′,6′-O-benzylidene-penta-N-benzyloxycarbonylparomomycin (2)

Sodium carbonate (55.0 g, 0.523 mol) and Cbz-Cl (20.00 mL, 0.139 mol)were added to paromomycin sulfate (30.00 g, 0.0271 mol) in water (500mL). After 35 hours under vigorous stirring, the water was decanted andthe white precipitate was washed with water twice. A solution oftriethylamine (97.00 mL, 0.697 mol) in methanol (600 mL) was added,followed by Cbz-Cl (25.00 mL, 0.174 mol). After 24 hours, dimethylamine(100 mL of a 40% aqueous solution) was added to quench the remainingCbz-Cl. The solvents were evaporated and the oil was washed with 3%methanol in ether twice and water. The resulting sticky solid was codistilled with pyridine (200 mL) three times and at ½ of the volume ofthe third co distillation, toluene (200 mL) was added and the solventswere evaporated to dryness. Another co-distillation with toluene (300mL) was done before heating the flask at 60° C. under 10 mm Hg vacuumfor 12 hours. Freshly distilled benzaldehyde (400 mL) was added to theresulting white solid and sonication was used to form a solution. To thestirred mixture was added 4 angstrom molecular sieves (15g) and formicacid (20.00 mL, 0.530 mol). After stirring for 12 hours at roomtemperature, the mixture was added dropwise to a stirred ice-coldsolution of saturated aqueous Na₂CO₃, extracted with ethyl acetate (3times), and the organic layer was washed with water, brine and driedover Na₂SO₄. The solvent was evaporated to dryness and excessbenzaldehyde was removed under vacuum to afford a crude solid, which waspurified by flash column chromatography over silica gel (3% MeOH/CH₂Cl₂)to obtain pure Compound 2 (23.89 g, 63%).

The spectroscopic analysis of the resulting material was consistent withdata reported in the literature for the identical material (HanessianS., Takamoto T., Massé R., Patil G.; Aminoglycoside antibiotics:Chemical conversion of neomycin B, paromomycin, and lividomycin B intobioactive pseudosaccharides; Can. J. Chem., 1978, 56, 1482).

Example 24′,6′-O-benzylidene-penta-N-benzyloxycarbonyl-5″-O-tert-butyldimethylsilylparomomycin (3)

The alcohol, Compound 2 (6.00 g, 4.367 mmol) dried by two codistillations with toluene was dissolved in CH₂Cl₂ (400 mL) and2,4,6-collidine (1.15 mL, 8.735 mmol) followed by TBDMSOTf (0.50 mL,2.184 mmol) were added at 0° C. After 18 hours, 0.6 equivalent ofTBDMSOTf was added and 6 hours later, some of the CH₂Cl₂ was evaporatedto a smaller volume for washing with HCl (0.5 M) twice and H₂O. Dryingwith Na₂SO₄ and purification by silica gel chromatography (2%MeOH/CH₂Cl₂) gave Compound 3 (4.861 g, 75%).

[α]_(D)+41.8° (c 0.9, CHCl₃); R_(f) 0.6 (CHCl₃:EtOAc:MeOH (20:5:3); ¹HNMR (300 MHz, CDCl₃) δ7.60-7.10 (m, 30H), 5.60-3.00 (m, 41H), 2.20 (m,1H), 1.30 (m, 1H), 0.83 (s, 9H), 0.01 (s, 6H); ESI m/z calcdC₇₆H₉₃N₅O₂₄Si 1487.60 found 1488.9.

Example 32″-O-allyl-4′,6′-O-benzylidene-penta-N-benzyloxycarbonyl-5″-O-tert-butyldimethylsilylparomomycin (4)

Compound 3 (2.10 g, 1.411 mmol) was co-distilled with toluene twice andthe residue dissolved in dry THF (70 mL) in a flask covered withaluminum foil. Allyl iodide (1.29 mL, 14.11 mmol) was added followed bythe dropwise addition of 0.5 M KHMDS solution in toluene (1.411 mL,0.706 mmol). The mixture was stirred for overnight at room temperature,then, 0.3 equivalents of KHMDS was added and 6 hours later the reactionmixture was quenched with an aqueous solution of NH₄Cl saturated (2 mL)and water. THF was evaporated and the aqueous layer was extracted withethyl acetate (3 times), and the organic layer was washed with a sodiumthiosulfate solution, brine and dried over Na₂SO₄. The solvent wasevaporated to dryness to afford a crude solid, which was purified bysilica gel flash chromatography (1.5% MeOH/CH₂Cl₂) providing thecorresponding allyl ether, Compound 4 (1.468 g, 68%).

[α]_(D)+22.2° (c 2.6, CHCl₃); R_(f) 0.7 (CHCl₃:EtOAc:MeOH (20:5:3); ¹HNMR (300 MHz, CDCl₃) δ7.60-7.10 (m, 30H), 6.30-3.00 (m, 44H), 2.20 (m,1H), 1.30 (m, 1H), 0.83 (s, 9H), 0.01 (s, 6H); ¹³C NMR (75 MHz, CDCl₃) δ157.7, 157.1, 156.5, 155.6, 137.2, 136.2, 135.7, 128.8, 128.5, 128.4,128.0, 127.9, 127.4, 126.3, 126.0, 101.5, 99.4, 85.2, 82.3, 81.4, 77.2,76.9, 76.6, 76.2, 74.2, 72.7, 69.5, 68.5, 67.3, 66.7, 63.5, 62.8, 56.5,52.7, 50.8, 40.1, 33.7, 25.8, 18.1, 14.1, −5.3, −5.5, −5.8; ESI m/zcalcd for C₇₉H₉₇N₅O₂₄Si 1527.63, found 1528.8.

Example 42″-O-allyl-3′,3′,4′-tri-O-benzoyl-4′,6′-O-benzylidene-penta-N-benzyloxycarbonyl-5″-O-tert-butyldimethylsilylparomomycin (5)

A solution containing Compound 4 (5.30 g, 3.46 mmol) andN,N-dimethyl-4-aminopyridine (100 mg) in dry pyridine (100 mL) wastreated with benzoyl chloride (3.017 mL, 34.641 mmol). The reactionmixture was stirred at room temperature for 36 hours water (5 mL) wasadded and after standing for 10 min, the solvent was removed undervacuum. The residue was dissolved in ethyl acetate, and the organiclayer was washed with NaHCO₃ saturated, 0.5 M HCl and water, dried overNa₂SO₄ and concentrated under vacuum. The crude product was purified bysilica gel flash column chromatography (1:1 EtOAc/hexane) to yieldCompound 5 (5.3 g, quantitative).

[α]_(C)+11.6° (c 2.5, CHCl₃); R_(f) 0.6 (1:1 EtOAc/hexane); ¹H NMR (300MHz, CDCl₃) δ 8.10-7.10 (m, 47H), 6.30-3.00 (m, 44H), 2.20 (m, 1H), 1.30(m, 1H), 0.83 (s, 9H), 0.01 (s, 6H); ¹³C NMR (75 MHz, CDCl₃) δ158.5,156.4, 138.0, 137.0, 136.9, 136.8, 136.5, 129.6, 129.5, 129.4, 129.2,129.1, 129.0, 128.8, 128.7, 128.4, 128.3, 128.2, 128.1, 127.0, 98.5,82.2, 78.1, 70.3, 70.2, 68.0, 67.8, 67.6, 67.4, 67.2, 26.6, 18.9; ESIm/z calcd for C₁₀₀H₁₀₉N₅O₂₇Si 1839.71 found 1840.9.

Example 53′,3′″,4′″-tri-O-benzoyl-4′,6′-O-benzylidene-penta-N-benzyloxycarbonyl-2″-O-methylenecarbonyl-5″-O-tert-butyldimethylsilylparomomycin (6)

The allyl ether derivative, Compound 5 (2.00 g, 1.086 mmol) in CH₂Cl₂(60 mL) was cooled at −78° C. and ozone was bubbled for 2 hours afterwhich excess ozone was removed by bubbling argon. The mixture wastreated with PPh₃ (427 mg, 1.629 mmol), warmed to room temperature andthe solvent was removed under vacuum. The crude solid was purified bysilica gel flash chromatography (2:3 EtOAc/hexane) to give the aldehyde,Compound 6 (1.627 g, 80%).

R_(f) 0.4 (1:1 EtOAc/hexane); ESI m/z C₉₉H₁₀₇N₅O₂₈Si 1841.69, found1842.9.

Example 6 General Procedure for Reductive Amination:

Compound R¹ R² 7a H

7b H

7c H

7d H

7e H

7f H

7g Me Me 7h

7i

7l H

7m H

7n H

7o H

7p H

7q H

7r H

7s H

7t H

7u H

7v H

7w H

7x H

7y H

7z H

7aa H

7ab H

7ac H

7ad H

7ae H

7af H

7ag

7ah H

7ai

7aj H

7ak H

To a mixture of Compound 6 (80.0 mg, 0.043 mmol) and the appropriateamine (0.129 mmol) in dry MeOH (3 mL) was added acetic acid (0.1 mL)followed by NaBH₃CN (1.0 M in THF, 60 μL). The mixture was stirred atroom temperature overnight. The solvents were removed under vacuum andthe crude solid was dissolved in ethyl acetate and washed with asolution of NaHCO₃ saturated and dried over Na₂SO₄. After evaporation ofthe solvents, the residue was purified by flash chromatography.

Compound 7a. 90% yield from 2-aminopyridine and compound 6 using thegeneral procedure above; silica gel flash chromatographyeluent:EtOAc:hexane (4:1); [α]_(D)+15.7° (c 1.3, CHCl₃); R_(f) 0.5(EtOAc); ESI m/z C₁₀₄H₁₁₃N₇O₂₇Si 1919.75, found 1920.8;

Compound 7b. 90% yield from 2-(aminomethyl)pyridine and compound 6 usingthe general procedure above; silica gel flash chromatography eluent: 3%MeOH/CH₂Cl₂; [α]_(D)+17.8° (c 0.9, CHCl₃); R_(f) 0.6 (5% MeOH/CH₂Cl₂);ESI m/z C₁₀₅H₁₁₅N₇O₂₇Si 1933.76, found 1934.8;

Compound 7c. 90% yield from N-1-(benzyloxycarbonyl)-1,3-diaminopropaneand compound 6 using the general procedure above; silica gel flashchromatography eluent: 3% MeOH/CH₂Cl₂; +12.7° (c 0.8, CHCl₃); R_(f) 0.5(5% MeOH/CH₂Cl₂); FAB m/z C₁₁₀H₁₂₃N₇O₂₉Si 2033.81, found 2036.1.

Compound 7d. 90% yield from N-1-(benzyloxycarbonyl)-1,2-diaminoethaneand compound 6 using the general procedure above; silica gel flashchromatography eluent: 3% MeOH/CH₂Cl₂; [α]_(D)+21.6.7° (c 1.7, CHCl₃);R_(f) 0.5 (5% MeOH/CH₂Cl₂); ESI m/z C₁₀₉H₁₂₁N₇O₂₉Si 2019.80, found2021.9;

Compound 7e. 90% yield from 2-aminomethylbenzimidazole and compound 6using the general procedure above (note: the benzylidene and the TBSwere often removed during the reductive amination); silica gel flashchromatography eluent: 7% MeOH/CH₂Cl₂; [α]_(D)+11.5° (c 1.1, CHCl₃);R_(f) 0.5 (10% MeOH/CH₂Cl₂); ESI m/z C₉₄H₉₈N₈O₂₇ 1770.65, found 1771.7;

Compound 7f. 90% yield from p-methylbenzylamine and compound 6 using thegeneral procedure above; silica gel flash chromatography eluent: 3%MeOH/CH₂Cl₂; [α]_(D)+8.9° (c 1.7, CHCl₃); R_(f) 0.6 (5% MeOH/CH₂Cl₂);ESI m/z C₁₀₇H₁₁₈N₆O₂₇Si 1946.78, found 1947.5.

Compound 7g. 90% yield from dimethylamine and compound 6 using thegeneral procedure above; silica gel flash chromatography eluent: 3%MeOH/CH₂Cl₂; [α]_(D)+28.3° (c 0.8, CHCl₃); R_(f) 0.6 (10% MeOH/CH₂Cl₂);ESI m/z C₁₀₁H₁₁₄N₆O₂₇Si 1870.75, found 1871.8;

Compound 7h. 90% yield from bis-[N-1-(benzyloxycarbonyl)aminoethyl]amineand compound 6 using the general procedure above; silica gel flashchromatography eluent: 3% MeOH/CH₂Cl₂; [α]_(D)+10.8° (c 1.5, CHCl₃);R_(f) 0.7 (5% MeOH/CH₂Cl₂); ESI m/z C₁₀₂H₁₁₆N₈O₃₃ 1980.76, found 1981.7;

Compound 7i. 90% yield from N-1-(benzyloxycarbonyl)piperazine andcompound 6 using the general procedure above; silica gel flashchromatography eluent: 3% MeOH/CH₂Cl₂; [α]_(D)+13.1° (c 1.2, CHCl₃);R_(f) 0.5 (5% MeOH/CH₂Cl₂); FAB m/z C₁₁₈H₁₂₈N₇O₃₀Si 2150.85, found2149.6.

Compound 7l. 88% yield from aniline and compound 6 using the generalprocedure above; ESI m/z C₁₀₅H₁₁₄N₆O₂₇Si 1920.14, found 1921.0; No ¹HNMR available.

Compound 7m. 84% yield from 3-aminoquinoline and compound 6 using thegeneral procedure above; ESI m/z C₁₀₈H₁₁₅N₇O₂₇Si 1971.18, found 1972.0

Compound 7n. 88% yield from cyclohexylamine and compound 6 using thegeneral procedure above; ESI m/z C₁₀₅H₁₂₀N₆O₂₇Si 1926.19, found 1927.0

Compound 7o. 92% yield from 3-(2-aminoethyl)pyridine and compound 6using the general procedure above; ESI m/z C₁₀₆H₁₁₇N₇O₂₇Si 1949.18,found 1950.3

Compound 7p. 74% yield from n-phenethylamine and compound 6 using thegeneral procedure above; ESI m/z C₁₀₇H₁₁₈N₆O₂₇Si 1948.19, found 1949.1

Compound 7q was prepared from benzylamine and compounds 6 and wassubsequently taken on directly to the next step without furthercharacterization.

Compound 7r was prepared from 3-aminophenol and compounds 6 and wassubsequently taken on directly to the next step without furthercharacterization.

Compound 7s was prepared fromN-2-(t-butoxycarbonylamino)-5-(aminomethyl)pyridine and compounds 6 andwas subsequently taken on directly to the next step without furthercharacterization.

Compound 7t was prepared fromN-2-(t-butoxycarbonylamino)-4-(aminomethyl)pyridine and compounds 6 andwas subsequently taken on directly to the next step without furthercharacterization.

Compound 7u. 90% yield from 2-aminopyridine and compound 6 using thegeneral procedure above; ESI m/z C₁₀₄H₁₁₃N₇O₂₇Si 1921.13, found 1921.0

Compound 7v was prepared from 3,3-dimethylaminopropane and compounds 6and was subsequently taken on directly to the next step without furthercharacterization.

Compound 7w was prepared from 1-amino-3-hydroxyadamantane and compounds6 and was subsequently taken on directly to the next step withoutfurther characterization.

Compound 7x. 85% yield from n-phenpropylamine and compound 6 using thegeneral procedure above; ESI m/z C₁₀₈H₁₂₀N₆O₂₇Si 1962.22, found 1963.3

Compound 7y was prepared from 1-amino-2-(2,4-dimethoxyphen-1-yl)ethaneand compounds 6 and was subsequently taken on directly to the next stepwithout further characterization.

Compound 7z was prepared from n-phenbutylamine and compound 6 and wassubsequently taken on directly to the next step without furthercharacterization.

Compound 7aa was prepared from (4-phenyl)phenethylamine and compound 6and was subsequently taken on directly to the next step without furthercharacterization.

Compound 7ab was prepared from 1-amino-2-(norborn-2-yl)ethane andcompound 6 and was subsequently taken on directly to the next stepwithout further characterization.

Compound 7ac was prepared from 2-aminonapthylene and compound 6 and wassubsequently taken on directly to the next step without furthercharacterization.

Compound 7ad was prepared from the amino-substituted cholesterol andcompound 6 and was subsequently taken on directly to the next stepwithout further characterization.

Compound 7ae was prepared from2-(2-Amino-ethyl)-benzo[de]isoquinoline-1,3-dione and compounds 6 andwas subsequently taken on directly to the next step without furthercharacterization.

Compound 7af was prepared from2-(3,5-Bis-trifluoromethyl-phenyl)-ethylamine and compound 6 and wassubsequently taken on directly to the next step without furthercharacterization.

Compound 7ag was prepared from Phenethyl-(3-phenyl-propyl)-amine andcompound 6 and was subsequently taken on directly to the next stepwithout further characterization.

Compound 7ah was prepared from 2-(4-Trifluoromethyl-phenyl)-ethylamineand compound 6 and was subsequently taken on directly to the next stepwithout further characterization.

Compound 7ai was prepared from dioctylamine and compound 6 and wassubsequently taken on directly to the next step without furthercharacterization.

Compound 7aj was prepared from 2-(4-Methoxyphenyl)ethylamine andcompound 6 and was subsequently taken on directly to the next stepwithout further characterization.

Compound 7ak was prepared from 2-(napthyl)ethylamine and compound 6 andwas subsequently taken on directly to the next step without furthercharacterization.

Example 7 General Procedure for Debenzoylation:

Compound R¹ R² 8a H

8b H

8c H

8d H

8e H

8f H

8g Me Me 8h

8i

8l H

8m H

8n H

8o H

8p H

8q H

8r H

8s H

8t H

8u H

8v H

8w H

8x H

8y H

8z H

8aa H

8ab H

8ac H

8ad H

8ae H

8af H

8ag

8ah H

8ai

8aj H

8ak H

The ester was treated with a catalytic amount of NaOMe in MeOH (1:1, 2mL, pH 9-10) and stirred at room temperature for overnight. The solutionwas cooled down to −78° C. and dry ice was added, solvent was removedunder vacuum and the residue was taken in CH₂Cl₂ and filtered overCelite. After removal of the solvent under vacuum the solid was purifiedby silica gel flash chromatography.

Compound 8a. 95% yield from compound 7a following the general procedure;silica gel flash chromatography eluent: 5% MeOH/CH₂Cl₂; [α]_(D)+8.9° (c1.4, MeOH); R_(f) 0.2 (5% MeOH/CH₂Cl₂); ESI m/z C₈₃H₁₀₁N₇O₂₄Si 1607.67,found 1630.8 (M+Na);

Compound 8b. 95% yield from compound 7b following the general procedure;silica gel flash chromatography eluent: 5% MeOH/CH₂Cl₂; [α]_(D)+10.3° (c1.1, MeOH); R_(f) 0.1 (5% MeOH/CH₂Cl₂); ESI m/z C₈₄H₁₀₃N₇O₂₄Si 1621.68,found 1644.8 (M+Na);

Compound 8c. 95% yield from compound 7c following the general procedure;silica gel flash chromatography eluent: 5% MeOH/CH₂Cl₂; R_(f) 0.1 (5%MeOH/CH₂Cl₂).

Compound 8d. 95% yield from compound 7d following the general procedure;silica gel flash chromatography eluent: 5% MeOH/CH₂Cl₂; R_(f) 0.1 (5%MeOH/CH₂Cl₂);

Compound 8e. 95% yield from compound 7e following the general procedure(the benzylidene and the TBS were removed during the reductiveamination); silica gel flash chromatography eluent: 10% MeOH/CH₂Cl₂;[α]_(D)+7.3° (c 1.6, MeOH); R_(f) 0.2 (10% MeOH/CH₂Cl₂); ESI m/zC₇₃H₈₆N₈O₂₄Si 1458.58, found 1459.7;

Compound 8f. 95% yield from compound 7f following the general procedure;silica gel flash chromatography eluent: 5% MeOH/CH₂Cl₂; [α]_(D)+11.3° (c0.8), MeOH)R_(f) 0.1 (5% MeOH/CH₂Cl₂). ESI m/z C₇₃H₈₈N₆O₂₄Si 1432.59,found 1433.4;

Compound 8g. 95% yield from compound 7g following the general procedure;silica gel flash chromatography eluent: 10% MeOH/CH₂Cl₂; [α]_(D)+11.6°(c 1.1, MeOH); R_(f) 0.4 (10% MeOH/CH₂Cl₂);

Compound 8i. 95% yield from compound 9i following the general procedure;silica gel flash chromatography eluent: 5% MeOH/CH₂Cl₂; [α]_(D)+17.6° (c0.4, MeOH)R_(f) 0.3 (5% MeOH/CH₂Cl₂). ESI m/z C₉₀H₁₁₂N₇O₂₆Si 1734.74found 1732.1.

Compound 8l. 82% yield from compound 7l following the general procedure;ESI m/z C₈₇H₁₀₂N₆O₂₄Si 1607.82, found 1608.9; ¹H NMR was taken and isconsistent with the structure.

Compound 8m. 79% yield from compound 7m following the general procedure;ESI m/z C₈₇H₁₀₃N₇O₂₄Si 1658.87, found 1659.9; ¹H NMR was taken and isconsistent with the structure.

Compound 8n. 80% yield from compound 7n following the general procedure;ESI m/z C₈₄H₁₀₈N₆O₂₄Si 1613.87, found 1614.9; ¹H NMR was taken and isconsistent with the structure.

Compound 8o. 86% yield from compound 7o following the general procedure;ESI m/z C₈₅H₁₀₅N₇O₂₄Si 1636.86, found 1637.2; ¹H NMR was taken and isconsistent with the structure.

Compound 8p. 82% yield from compound 7p following the general procedure;ESI m/z C₈₆H₁₀₆N₆O₂₄Si 1635.87, found 1636.0; ¹H NMR was taken and isconsistent with the structure.

Compound 8q. 78% yield from compound 7q following the general procedure;ESI m/z C₈₅H₁₀₄N₆O₂₄Si 1621.85, found 1622.1; ¹H NMR was taken and isconsistent with the structure.

Compound 8r. 78% yield from compound 7r following the general procedure;ESI m/z C₈₄H₁₀₂N₆O₂₅Si 1623.82, found 1623.8; ¹H NMR was taken and isconsistent with the structure.

Compound 8s. 81% yield from compound 7s following the general procedure;ESI m/z C₈₉H₁₁₂N₈O₂₆Si 1737.97, found 1738.9; ¹H NMR was taken and isconsistent with the structure.

Compound 8t. 86% yield from compound 7t following the general procedure;ESI m/z C₈₉H₁₁₂N₈O₂₆Si 1737.97, found 1738.2; ¹H NMR was taken and isconsistent with the structure.

Compound 8u. 85% yield from compound 7u following the general procedure;ESI m/z C₈₃H₁₀₁N₇O₂₄Si 1608.81, found 1608.8; ¹H NMR was taken and isconsistent with the structure.

Compound 8v. 72% yield from compound 7v following the general procedure;ESI m/z C₈₄H₁₁₀N₆O₂₄Si 1615.88, found 1615.8; ¹H NMR was taken and isconsistent with the structure.

Compound 8w. 91% yield from compound 7w following the general procedure;ESI m/z C₈₈H₁₁₂N₆O₂₅Si 1681.94, found 1681.6; ¹H NMR was taken and isconsistent with the structure.

Compound 8x. 90% yield from compound 7x following the general procedure;ESI m/z C₈₇H₁₀₈N₆O₂₄Si 1649.90, found 1671.9 (M+Na); ¹H NMR was takenand is consistent with the structure.

Compound 8y. 84% yield from compound 7y following the general procedure;ESI m/z C₈₈H₁₁₀N₆O₂₆Si 1695.93, found 1695.9; ¹H NMR was taken and isconsistent with the structure.

Compound 8z. 95% yield from compound 7z following the general procedure;ESI m/z C₈₈H₁₁₀N₆O₂₄Si 1663.93, found 1686.1 (M+Na); ¹H NMR was takenand is consistent with the structure.

Compound 8aa. 81% yield from compound 7aa following the generalprocedure; ESI m/z C₉₂H₁₁₀N₆O₂₄Si 1711.97, found 1711.9; ¹H NMR wastaken and is consistent with the structure.

Compound 8ab. 73% yield from compound 7ab following the generalprocedure; ESI m/z C₈₇H₁₁₂N₆O₂₄Si 1652.75, found 1653.7; ¹H NMR wastaken and is consistent with the structure.

Compound 8ac. 80% yield from compound 7ac following the generalprocedure; ESI m/z C₈₈H₁₀₈N₆O₂₄Si 1661.91, found 1661.6; ¹H NMR wastaken and is consistent with the structure.

Compound 8ad. 87% yield from compound 7ad following the generalprocedure; ESI m/z C₁₀₅H₁₄₄N₆O₂₄Si 1902.38, found 1902.2; ¹H NMR wastaken and is consistent with the structure.

Compound 8ae. 70% yield from compound 7e following the generalprocedure; ESI m/z C₉₂H₁₀₇N₇O₂₆Si 1754.95, found 1755.7; ¹H NMR wastaken and is consistent with the structure.

Compound 8af. 85% yield from compound 7af following the generalprocedure; ESI m/z C₈₅H₁₀₄F₆N₆O₂₄Si 1771.87, found 1771.5; ¹H NMR wastaken and is consistent with the structure.

Compound 8ag. 88% yield from compound 7ag following the generalprocedure; ESI m/z C₉₅H₁₁₆N₆O₂₄Si 1754.05, found 1756.4; ¹H NMR wastaken and is consistent with the structure.

Compound 8ah. 94% yield from compound 7ah following the generalprocedure; ESI m/z C₈₇H₁₀₅F₃N₆O₂₄Si 1703.87, found 1703.5; ¹H NMR wastaken and is consistent with the structure.

Compound 8ai. 95% yield from compound 7ai following the generalprocedure; ESI m/z C₉₄H₁₃₀N₆O₂₄Si 1756.15, found 1756.3; ¹H NMR wastaken and is consistent with the structure.

Compound 8aj. 83% yield from compound 7aj following the generalprocedure; ESI m/z C₈₇H₁₀₈N₆O₂₅Si 1665.9, found 1665.6; ¹H NMR was takenand is consistent with the structure.

Compound 8ak was prepared from compound 7ak following the generalprocedure and was subsequently taken on directly to the next stepwithout further characterization.

Example 8 General Procedure for Final Deprotection:

Compound R¹ R² 9a H

9b H

9c H

9d H

9e H

9f H H 9g Me Me 9h

9i

9j H

9k H

9l H

9m H

9n H

9o H

9p H

9q H

9r H

9s H

9t H

9u H

9v H

9w H

9x H

9y H

9z H

9aa H

9ab H

9ac H

9ad H

9ae H

9af H

9ag

9ah H

9ai

9aj H

9ak H

The appropriate substrate was dissolved in 80% aqueous acetic acid (3mL) and heated at 60° C. for 3 hours The solution was cooled down toroom temperature and a catalytic amount of 20% palladium hydroxide oncarbon was added and the suspension was stirred at room temperatureunder an atmosphere of hydrogen (hydrogen balloon) until the conversionof the starting material into the product was completed as indicated byMS analysis. The mixture was filtered through a layer of Celite oncotton, concentrated under vacuum, washed with CH₂Cl₂ and lyophilized toafford floppy white solids.

Compound 9a. Quantitative yield from compound 8a following the generalprocedure; [α]_(D)+6.8° (c 0.4, H₂O); ¹H NMR (400 MHz, D₂O) δ 8.00-7.70(m, 2H), 7.60-7.40 (m, 2H), 5.70 (m, 1H), 5.33 (m, 1H), 5.11 (m, 1H),4.50 (m, 1H), 4.20-4.00 (m, 4H), 3.85-3.50 (m, 13H), 3.40-3.15 (m, 8H),2.37 (m, 1H), 1.79 (s, 18H), 1.70 (m, 1H); ¹³C NMR (125 MHz, D₂O) δ181.4, 132.3, 131.6, 129.5, 129.2, 128.7, 127.1, 126.8, 108.8, 96.2,95.3, 85.3, 81.6, 81.0, 78.0, 74.1, 73.1, 70.7, 69.6, 69.3, 68.0, 67.7,60.7, 60.3, 54.2, 51.5, 50.3, 49.2, 43.0, 40.7, 29.2, 23.5; ESI m/zC₃₀H₅₃N₇O₁₄ 735.37, found 736.5;

Compound 9b. Quantitative yield from compound 8b following the generalprocedure; [α]_(D)+5.4° (c 0.6, H₂O); ¹H NMR (400 MHz, D₂O) δ 7.70-7.30(m, 4H), 5.71 (m, 1H), 5.38 (m, 1H), 5.16 (m, 1H), 4.55 (m, 1H),4.20-4.00 (m, 4H), 3.95-3.50 (m, 15H), 3.45-3.15 (m, 8H), 2.32 (m, 1H),1.81 (s, 18H), 1.65-1.40 (m, 1H); ¹³C NMR (125 MHz, D₂O) δ 181.4, 150.5,140.0, 133.9, 132.8, 129.8, 129.1, 128.9, 128.2, 125.5, 109.0, 96.6,95.7, 85.7, 81.4, 78.5, 74.3, 73.7, 71.1, 69.9, 68.5, 68.1, 61.0, 60.1,54.6, 51.6, 50.8, 49.6, 46.6, 41.1, 31.8, 29.7, 23.5; ESI m/zC₃₁H₅₅N₇O₁₄ 749.38, found 750.4;

Compound 9c. Quantitative yield from compound 8c following the generalprocedure; [α]_(D)+5.7° (c 0.4, H₂O); ¹H NMR (400 MHz, D₂O) δ 5.72 (m,1H), 5.44 (m, 1H), 5.21 (m, 1H), 4.59 (m, 1H), 4.20-4.00 (m, 4H),3.95-3.50 (m, 13H), 3.45-2.7 (m, 14H), 2.26 (m, 1H), 1.87 (s, 21H), 1.59(m, 1H); ¹³C NMR (125 MHz, D₂O) δ 182.2, 108.9, 96.8, 96.0, 86.0, 81.8,79.8, 74.5, 74.3, 71.3, 71.2, 70.1, 68.6, 68.2, 67.5, 61.0, 54.8, 51.8,51.1, 50.3, 49.8, 48.8, 45.5, 43.7, 41.1, 37.3, 31.1, 27.4, 24.5, 24;ESI m/z C₂₈H₅₇N₇O₁₄ 715.40, found 716.4;

Compound 9d. Quantitative yield from compound 8d following the generalprocedure; [α]_(D)+8.1° (c 0.6, H₂O); ¹H NMR (400 MHz, D₂O) δ 5.75 (m,1H), 5.44 (m, 1H), 5.20 (m, 1H), 4.30-4.00 (m, 4H), 3.85-3.50 (m, 13H),3.40-3.15 (m, 8H), 3.00-2.55 (m, 4H) 2.31 (m, 1H), 1.91 (s, 21H), 1.63(m, 1H); ESI m/z C₂₇H₅₅N₇O₁₄ 701.38, found 702.6;

Compound 9e. Quantitative yield from compound 8e following the generalprocedure; [α]_(D)+8.6° (c 0.7, H₂O); ¹H NMR (400 MHz, D₂O) δ 7.80-7.40(m, 4H), 5.81 (m, 1H), 5.44 (m, 1H), 5.24 (m, 1H), 4.35-4.10 (m, 4H),3.95-3.50 (m, 14H), 3.45-3.15 (m, 8H), 2.42 (m, 1H), 1.91 (s, 18H), 1.61(m, 1H); ESI m/z C₃₃H₅₆N₈O₁₄ 788.39, found 789.5;

Compound 9f. Quantitative yield from compound 8f following the generalprocedure; [α]_(D)+10.6° (c 0.7, H₂O); ¹H NMR (400 MHz, D₂O) δ 5.78 (m,1H), 5.46 (m, 1H), 5.26 (m, 1H), 4.30-4.00 (m, 6H, 3.95-3.50 (m, 14H),3.45-3.00 (m, 6H), 2.35 (m, 1H), 1.91 (s, 21H), 1.71 (m, 1H); ESI m/zC₂₅H₅₀N₆O₁₄ 658.33, found 659.4;

Compound 9g. Quantitative yield from compound 8g following the generalprocedure; [α]_(D)+7.3° (c 0.6, H₂O); ¹H NMR (400 MHz, D₂O) δ 5.76 (m,1H), 5.46 (m, 1H), 5.26 (m, 1H), 4.62 (m, 1H), 4.41-4.04 (m, 5H,3.90-3.50 (m, 14H), 3.45-3.20 (m, 6H), 2.9 (s, 6H) 2.33 (m, 1H), 1.88(s, 18H), 1.70 (m, 1H); ¹³C NMR (125 MHz, D₂O) δ 182.0, 108.8, 96.7,95.6, 85.8, 81.4, 81.2, 78.9, 74.3, 74.2, 73.9, 71.2, 69.9, 69.8, 68.5,68.0, 64.9, 60.9, 59.9, 57.5, 54.7, 51.7, 50.9, 49.6, 43.6 (2C), 41.1,30.2, 23.9; ESI m/z C₂₇H₅₄N₆O₁₄ 686.4, found 687.4;

Compound 9h. Quantitative yield from compound 8h following the generalprocedure; [α]_(D)+21.5° (c 0.6, H₂O); ¹H NMR (400 MHz, D₂O) δ 5.55 (m,1H), 5.16 (m, 1H), 5.08 (m, 1H), 4.49 (m, 1H), 4.30-4.00 (m, 5H,3.95-3.40 (m, 14H), 3.45-3.15 (m, 6H), 2.58 (m, 8H) 2.18 (m, 1H), 1.92(s, 24H), 1.30 (m, 1H); ESI m/z C₂₉H₆₀N₈O₁₄ 744.42, found 745.6;

Compound 9i. Quantitative yield from compound 8i following the generalprocedure; [α]_(D)+14.5° (c 0.7, H₂O); NMR (400 MHz, D₂O) δ 5.70 (m,1H), 5.35 (m, 1H), 5.12 (m, 1H), 4.49 (m, 1H), 4.30-4.00 (m, 5H,3.95-3.40 (m, 14H), 3.45-3.05 (m, 10H), 2.68 (m, 4H), 2.26 (m, 1H), 1.87(s, 21H), 1.62 (m, 1H); ¹³C NMR (125 MHz, D₂O) δ 181.6, 108.9, 96.6,95.9, 87.5, 81.9, 81.6, 78.5, 74.6, 74.5, 73.7, 71.2, 70.0, 69.8, 68.5,68.1, 68.0, 61.0, 60.6, 57.2, 54.7, 51.8, 51.1, 50.8, 50.2 (2), 49.7,43.6 (2C), 41.1, 31.1, 23.6; ESI m/z C₂₉H₅₈N₇O₁₄ 728.40, found 728.3;

Compound 9j. Prepared by extened hydrogenation via 9a. quantitative;[α]_(D)+7.8° (c 1.0, H₂O); ¹H NMR (400 MHz, D₂O) δ 5.66 (m, 1H), 5.30(m, 1H), 5.11 (m, 1H), 4.46 (m, 1H), 4.20-4.00 (m, 5H, 3.95-3.50 (m,14H), 3.40-2.95 (m, 11H), 2.37 (m, 1H), 2.1-1.9 (m, 4H) 1.79 (s, 18H),1.70 (m, 1H); ¹³C NMR (125 MHz, D₂O) δ 181.0, 108.9, 96.6, 95.7, 90.9,85.5, 81.5, 77.7, 74.5, 74.3, 73.3, 71.1, 69.9, 69.5, 68.4, 68.3, 68.0,61.0, 54.5, 52.4, 51.5, 50.6, 49.4, 44.7, 44.2, 41.0, 40.1, 34.5, 28.9,23.3 20.9, 20.2; ESI m/z C₃₀H₅₉N₇O₁₄ 741.41, found 742.7;

Compound 9k. Prepared by extened hydrogenation via 9b. quantitative;[α]_(D)+12.4° (c 1.1, H₂O); ¹H NMR (400 MHz, D₂O) δ, 5.67 (m, 1H), 5.32(m, 1H), 5.25 (m, 1H), 4.48 (m, 1H), 4.20-4.00 (m, 5H), 3.95-3.30 (m,18H), 3.30-3.00 (m, 12H), 2.21 (m, 1H), 1.81 (s, 21H), 1.62 (m, 1H); ¹³CNMR (125 MHz, D₂O) δ 180.3, 108.5, 96.2, 95.2, 85.0, 81.1, 80.8, 77.2,74.1 (2C), 72.9, 70.7, 69.4, 69.1, 67.9, 67.5, 60.5, 60.1, 54.1, 51.1,50.3, 50.2, 49.1, 48.9, 46.1, 44.2, 44.1, 40.6, 30.3, 28.6, 22.75, 22.1,21.4, 18.2; ESI m/z C₃₁H₆₁N₇O₁₄ 755.42, found 756.7.

Compound 9l. 80% yield from compound 8l following the general procedure;ESI m/z C₃₁H₅₄N₆O₁₄ 734.79, found 735.5; ¹H NMR is consistent with thestructure.

Compound 9m. 85% yield from compound 8m following the general procedure;ESI m/z C₃₄H₅₅N₇O₁₄ 785.84, found 786.5; ¹H NMR is consistent with thestructure.

Compound 9n. 85% yield from compound 8n following the general procedure;ESI m/z C₃₁H₆₀N₆O₁₄ 740.84, found 741.5; ¹H NMR is consistent with thestructure.

Compound 9o. 85% yield from compound 8o following the general procedure;ESI m/z C₃₂H₅₇N₇O₁₄ 763.83, found 764.7; ¹H NMR is consistent with thestructure.

Compound 9p. 80% yield from compound 8p following the general procedure;ESI m/z C₃₃H₅₈N₆O₁₄ 762.85, found 763.6; ¹H NMR is consistent with thestructure.

Compound 9q. 85% yield from compound 8q following the general procedure;ESI m/z C₃₂H₅₆N₆O₁₄ 748.82, found 749.6; ¹H NMR is consistent with thestructure.

Compound 9r. 85% yield from compound 8r following the general procedure;ESI m/z C₃₁H₅₄N₆O₁₅ 750.79, found 751.6; ¹H NMR is consistent with thestructure.

Compound 9s. 60% yield from compound 8s following the general procedure;ESI m/z C₃₁H₅₆N₈O₁₄ 764.82, found 765.6; ¹H NMR is consistent with thestructure.

Compound 9t. 65% yield from compound 8t following the general procedure;ESI m/z C₃₁H₅₆N₈O₁₄ 764.82, found 765.6; ¹H NMR is consistent with thestructure.

Compound 9u. 75% yield from compound 8u following the general procedure;ESI m/z C₃₀H₅₃N₇O₁₄ 735.78, found 736.5; ¹H NMR is consistent with thestructure.

Compound 9v. 80% yield from compound 8v following the general procedure;ESI m/z C₃₁H₆₂N₆O₁₄ 742.86, found 743.4; ¹H NMR is consistent with thestructure.

Compound 9w. 80% yield from compound 8w following the general procedure;ESI m/z C₃₅H₆₄N₆O₁₅ 808.91, found 809.4; ¹H NMR is consistent with thestructure.

Compound 9x. 90% yield from compound 8x following the general procedure;ESI m/z C₃₄H₆₀N₆O₁₄ 776.87, found 777.6; ¹H NMR is consistent with thestructure.

Compound 9y. 90% yield from compound 8y following the general procedure;ESI m/z C₃₅H₆₂N₆O₁₆ 822.90, found 823.5; ¹H NMR is consistent with thestructure.

Compound 9z. 90% yield from compound 8z following the general procedure;ESI m/z C₃₅H₆₂N₆O₁₄ 790.90, found 791.7; ¹H NMR is consistent with thestructure.

Compound 9aa. 85% yield from compound 8aa following the generalprocedure; ESI m/z C₃₉H₆₂N₆O₁₄ 838.94, found 839.5; ¹H NMR is consistentwith the structure.

Compound 9ab. 80% yield from compound 8ab following the generalprocedure; ESI m/z C₃₄H₆₄N₆O₁₄ 780.90, found 781.5; ¹H NMR is consistentwith the structure.

Compound 9ac. 90% yield from compound 8ac following the generalprocedure; ESI m/z C₃₅H₆₀N₆O₁₄ 788.88, found 789.5; ¹H NMR is consistentwith the structure.

Compound 9ad. 80% yield from compound 8ad following the generalprocedure; ESI m/z C₅₂H₉₆N₆O₁₄ 1029.35, found 1029.7; ¹H NMR isconsistent with the structure.

Compound 9ae. 75% yield from compound 8ae following the generalprocedure; ESI m/z C₃₉H₅₉N₇O₁₆ 881.92, found 882.5; ¹H NMR is consistentwith the structure.

Compound 9af. 90% yield from compound 8af following the generalprocedure; ESI m/z C₃₅H₅₆F₆N₆O₁₄ 898.84, found 899.4; ¹H NMR isconsistent with the structure.

Compound 9ag. 90% yield from compound 8ag following the generalprocedure; ESI m/z C₄₈H₆₈N₆O₁₄ 881.02, found 883.8; ¹H NMR is consistentwith the structure.

Compound 9ah. 85% yield from compound 8ah following the generalprocedure; ESI m/z C₃₄H₅₇F₃N₆O₁₄ 830.84, found 831.5; ¹H NMR isconsistent with the structure.

Compound 9ai. 80% yield from compound 8ai following the generalprocedure; ESI m/z C₄₁H₈₂N₆O₁₄ 883.12, found 883.9; ¹H NMR is consistentwith the structure.

Compound 9aj. 90% yield from compound 8aj following the generalprocedure; ESI m/z C₃₄H₆₀N₆O₁₅ 792.87, found 793.7; ¹H NMR is consistentwith the structure.

Compound 9ak was prepared from compound 8ak following the generalprocedure.

Example 9 Preparation of Compounds 10 and 11

Compound 7p is treated with the appropriate acyl chloride (1.2 equiv)and then deprotected according to the general procedure to give 10(benzoyl chloride) and 11 (acetyl chloride).

Compound 10. 75% yield from compound 7p and benzoyl chloride followingthe general procedure; ESI m/z C₄₀H₆₂N₆O₁₅ 866.97, found 867.5; ¹H NMRis consistent with the structure.

Compound 11. 80% yield from compound 7p and acetyl chloride followingthe general procedure; ESI m/z C₃₅H₆₀N₆O₁₅ 804.88, found 806.3; ¹H NMRis consistent with the structure.

Example 10 Preparation of C2″-Alkoxy Ether Paromomycin

Compound 6 is treated with 5-10 equivalents of sodium borohydride inmethanol, and then deprotected according to the general procedure togive compound 12a.

Compound 12a. 80% yield; ESI m/z C₂₅H₄₉N₅O₁₅ 659.68, found 660.51; ¹HNMR is consistent with the structure.

Compound 12b was prepared according to the above reaction scheme.Reduction of 6 led to the first inteiniediate. Standard alkylation ofthe first intermediate with cinnamyl bromide gave the expected secondintermediate, which upon deprotection afforded 12b in which the phenylring had undergone overreduction to a cyclohexyl moiety.

Example 11 Preparation of Compound 13

Compound 6 is treated with 1-2 equivalents of phenylmagnesiumbromide ordiphenyl zinc in THF, and then deprotected according to the generalprocedure to give compound 13.

Compound 13. 65% yield; ESI m/z C₃₁H₅₃N₅O₁₅ 735.78, found 736.8; ¹H NMRis consistent with the structure.

Example 12 Preparation of 2″-Alkyl/Aryl Ether Paromomycin

Compound 3 (2.10 g, 1.411 mmol) was dissolved in dry THF (70 mL) andphenethyl chloride (10 equiv) was added followed by the dropwiseaddition of 0.5 M KHMDS solution in toluene (1.411 mL, 0.706 mmol). Themixture was stirred for overnight at room temperature, and thendeprotected according to the general procedures to provide phenethylether 14a.

Compound 14a. 85% yield; ESI m/z C₃₁H₅₃N₅O₁₄ 719.78, found 720.9; ¹H NMRis consistent with the structure.

Compound 14b was prepared according to the above reaction scheme. Directalkylation of Compound 3 with cinnamyl bromide in the presence of KHMDSand Bu₄NI at 0° C. gave the intermediate product in 70% yield.Alternatively, the intermediate product could also be obtained byperforming a cross-metathesis reaction of Compound 4 with styrene in thepresence of the Grubbs second generation catalyst in 75% yield (seeScholl, M.; Ding, S.; Lee, C. W.; Grubbs, R. H. Org. Lett. 1999, 1,953). Cleavage of the benzylidene acetal as well as the OTBS etherfollowed by catalytic hydrogenolysis gave Compound 14b.

Compound 14c was prepared according to the above reaction scheme. AWittig reaction of 6 gave the intermediate as a mixture of isomericolefins. Deprotection and hydrogenation afforded the 5-phenylpentylether analogue 14c.

Example 13 Preparation of N-Protected Paromomycin

The exocyclic amino groups of Paromomycin were converted into thecorresponding azido groups according to the procedure of Wong(Greenberg, W. A.; Priestley, E. S.; Sears, P. S.; Alper, P. B.;Rosenbohm, C. et al. Design and Synthesis of New AminoglycosideAntibiotics Containing Neamine as an Optimal Core Structure: Correlationof Antibiotic Activity with in Vitro Inhibition of Translation. J. Am.Chem. Soc. 1999, 121, 6527-6541) using paromomycin instead of neomycin.

¹H NMR (300 MHz, DMSO) δ 1.36 (q, J=12 Hz, 1H), δ 1.99-2.06 (m, 1H) δ3.37-3.73 (m, 1H) δ 2.97-3.02 (m, 1H), δ 3.19-3.27 (m, 1H), δ 3.37-3.73(m, 15H), δ 3.88-3.95 (m, 2H), δ 4.16-4.25 (m, 2H), δ 4.44 (t, J=5.7 Hz,1H) δ 4.75 (t, J=4.8 Hz, 1H), δ 4.93 (d, J=5.2 Hz, 1H), δ 5.03 (d, J=1.6Hz, 1H), δ 5.15 (d, J=5.1 Hz, 1H) δ 5.22 (d, J=4.6 Hz, 1H), δ 5.28 (s,1H), δ 5.39 (d, J=5.7 Hz, 1H), δ 5.59 (t, J=4.8 Hz, 2H), δ 5.67 (d,J=3.7 Hz, 1H); ¹³C NMR δ 106.97, 97.64, 95.89, 83.22, 81.67, 75.60,74.66, 74.13, 72.98, 72.80, 70.30, 70.00, 69.81, 66.99, 63.0261.50,60.40, 59.85, 59.66, 59.21, 50.77, 31.46 LCMS m/z 768.0 (M+Na), (>99%purity).

Example 14

Selective Protection of the 6′-Position with Tips

To an oven dried 50.0 mL bottom flask equipped with magnetic stirrer wasadded per-azidoparomomycin from the above reaction (2.63 g, 3.5 mmol),4-DMAP (1.25 g, 10.2 mmol) and anhydrous DMF (28.0 mL). The resultingclear solution was cooled to 0° C. in ice-bath while stirring undernitrogen. Triisopropylsilylchloride (0.89 mL, 42.3 mmol) was addeddropwise to the stirred reaction mixture via syringe. The reaction wascontinued stirred for two hours maintaining the temperature at 0° C. Thereaction mixture was then partitioned between ethyl acetate and 10%aqueous NaHCO₃ solution. The organic layer was separated and washed withsaturated brine solution and dried over Na₂SO₄, filtered and evaporatedto dryness to afforded clear oil. The product was obtained afterpurification by flash chromatography (1.57 g, 50% yield) using gradientsof CHCl₃/MeOH (97:3).

¹H NMR (300 MHz, DMSO) δ 1.36 (q, J=12 Hz, 1H), δ 1.90-1.22 (m, 21H) δ2.06-2.10 (m, 1H) δ 2.97-3.03 (m, 7H), δ 3.08-3.98 (m, 13H), δ 4.15 (s,2H), δ 4.6 (t, J=60.4 Hz, 1H), δ 4.94 (d, J=5.0 Hz, 1H), δ 4.99-5.03 (m,1H), δ 5.14 (d, J=3.73 Hz, 1H), δ 5.20 (d, J=4.6 Hz, 1H), δ 5.27 (s,1H), δ 5.44 (d, J=5.5 Hz, 1H), δ 5.59 (d, J=3.90 Hz, 1H), δ 5.68 (d,J=6.2 1H), δ 5.79 (d, J=3.73 Hz. 1H), δ 6.62 (dd, J=5.09, 1.5 Hz, 2H), δ8.10 (d, J=6.56 Hz, 1H); ¹³C NMR δ 154.19, 148.11, 108.18, 106.63,97.56, 95.38, 83.08, 81.62, 75.87, 75.52, 74.00, 73.79, 73.10, 72.76,70.41, 70.26, 69.76, 66.96, 63.31, 62.91, 62.20, 59.79, 59.58, 59.15,50.77, 38.64, 31.72, 17.81, 17.79, 11.37, 0.00 LCMS m/z 924 (M+Na),(>99% purity).

Example 15 Benzyl Protection of Hydroxyl Groups

To a 50.0 mL bottom flask equipped with magnetic stirrer was added thetips protected compound from the previous example (3.77 g, 4.18 mmol)dissolved in anhydrous DMF (20.0 mL). The resulting clear solution wascooled to 0° C. in ice-bath while stirring under nitrogen. 60% NaH (2.34g, 58.5 mmol) was then added slowly and stirred for 20 minutes. BnBr(4.97 mL, 41.87 mmol) was added dropwise to the stirred reaction mixturevia syringe. Temperature of 0° C. was maintained for 1 h followed by 3hat room temperature. The reaction was then cooled at 0° C. and quenchedwith saturated NaHCO₃ solution (2.0 mL) dropwise. The reaction mixturewas then partitioned between DCM and 10% aqueous NaHCO₃ solution. Theorganic layer was separated and washed with saturated brine solution anddried over Na₂SO₄, filter and evaporated to dryness to afforded clearoil which was purified by silica gel chromatography using gradients ofHexane/EtOA (9:1) to afford the title compound (6.02 g, 93% yield) whichwas used as is in the next step.

Example 16 Selective Deprotection of the 6′-Position of Perbenzylated6′-O-Tips-Perazido-Paromomycin and Oxidation to the Aldehyde

To a 50.0 mL bottom flask equipped with magnetic stirrer was added thebenzyl protected 6′-O-Tips-perazidoparomomycin (6.0 g, 3.92 mmol)dissolved in anhydrous THF (20 mL). The resulting clear solution wascooled to 0° C. in ice-bath while stirring under nitrogen. 1.0M TBAF•THF(8.63 mL, 7.84 mmol) was added dropwise to the stirred reaction mixturevia syringe and the reaction was then allowed to proceed at roomtemperature. The reaction was quenched with saturated NH₄CO₃ solution(30.0 mL), extracted with EtOAc and evaporated to dryness to affordedthe product as a yellow oil which could be purified by silica gelchromatography using gradients of Hexane/EtOA (8:2) to afforded thetitle compound (5.4 g, 83% yield) as a white foam. This product (470 mg)was treated with IBX in DMSO (1.2 mL) and THF (1.0 mL) at roomtemperature for 2.5 hours. At that time, DCM (15 mL) and H₂O (10 mL)were added and the aqueous layer was separated and extracted twice more(15 mL). The combined organic layers were dried (Na₂SO₄), filtered andevaporated to give crude product which could be purified by silica gelchromatography using gradients of Hexane/EtOA (7:3) to afford the titlecompound (409 mg, 50% yield).

General Procedure for Reductive Amination and Deprotection

The crude aldehyde (36:moles) was dissolved in dry MeOH (2 mL) and dryTHF (1 mL). To this solution was added the appropriate amine (5equivalents) in MeOH (2 mL) with the pH adjusted to 5 with AcOH. NaCNBH₃(4 equivalents) was then added and the mixture was allowed to stir for16 hours, at which time the reaction was quenched with NaHCO₃. Thereaction was evaporated to dryness, and then the crude mixture waspartitioned between DCM and 10% aqueous NaHCO₃ solution. The organiclayer was separated and washed with saturated brine solution and driedover Na₂SO₄, filter and evaporated to dryness to afforded clear oilwhich was purified by silica gel chromatography using gradients ofDCM:MeOH (96:4) to afford the protected amine, which was used as is inthe next step. To the protected amine was added 2 mL of EtOH, Raneynickel (25-50 mg) and hydrazine (7-14 equivalents). After the reactionhad gone to completion as determined by LCMS, the reaction was filteredand evaporated to give the crude perbenzylated product. This was treatedwith hydrogen (1 atm), palladium (II) hydroxide (2.5 mg) in AcOH (1 mL)and THF (1 mL) to give, after 24 hours, the title compound 15 afterlyophilization.

Example 18 Preparation of Compound 15a

Using 4M dimethylamine in methanol in the general procedure above gavethe title compound. LCMS m/z 643 (M+H), (>95% purity). ¹H NMR wasconsistent with the structure.

R═N(CH₃)₂, see Example 17.

Example 19 Preparation of Compound 15b

Using 1,3-diaminopropane in the general procedure above gave the titlecompound. LCMS m/z 672 (M+H), (>95% purity). ¹H NMR was consistent withthe structure.

R═N(H)(CH₂)₃NH₂ see Example 17.

Example 20 Preparation of Compound 15c

Using morpholine in the general procedure above gave the title compound.LCMS m/z 685 (M+H), (>95% purity). ¹H NMR was consistent with thestructure.

R=

see Example 17. Example 21 Preparation of Compound 15d

Using N-Boc-hydrazine in the general procedure above gave the titlecompound. LCMS m/z 730 (M+H), (>95% purity). ¹H NMR was consistent withthe structure.

R═N(H)N(H)—BOC, see Example 17.

Example 22 Preparation of Compound 15e

Using 2.0 M methylamine in methanol in the general procedure above gavethe title compound. LCMS m/z 629 (M+H), (>95% purity). ¹H NMR wasconsistent with the structure.

R═N(H)CH₃, see Example 17.

Example 23 Preparation of Compound 15f

Using 1,4-diaminobutane in the general procedure above gave the titlecompound. LCMS m/z 686 (M+H), (>95% purity). ¹H NMR was consistent withthe structure.

R═N(H)(CH₂)₄NH₂ see Example 17.

Example 24 Preparation of Compound 15g

Using p-Methylphenethylamine in the general procedure above gave thetitle compound. LCMS m/z 733 (M+H), (>95% purity). ¹H NMR was consistentwith the structure.

R=

see Example 17. Example 25 Preparation of Compound 15h

Using isopropylamine in the general procedure above gave the titlecompound. LCMS m/z 657 (M+H), (>95% purity). ¹H NMR was consistent withthe structure.

R═N(H)C(H)(CH₃)₂, see Example 17.

Example 26 Preparation of Compound 15i

Using hydrazine in the general procedure above gave the title compound.This compound can also be prepared from the protected hydrazinylcompound of Example 21. LCMS m/z 630 (M+H), (>95% purity). ¹H NMR wasconsistent with the structure.

R═N(H)NH₂, see Example 17.

Example 27 Preparation of Compound 15j

Using phenethylamine in the general procedure above gave the titlecompound. LCMS m/z 719 (M+H), (>95% purity). ¹H NMR was consistent withthe structure.

R═N(H)(CH₂)₂Ph, see Example 17.

Example 28 Preparation of Compound 15k

Using N-methyl-phenethylamine in the general procedure above gave thetitle compound. LCMS m/z 733 (M+H), (>95% purity). ¹H NMR was consistentwith the structure.

R═N(CH₃)(CH₂)₂Ph, see Example 17.

Example 29 Preparation of Compound 15l

Using phenpropylamine in the general procedure above gave the titlecompound. LCMS m/z 733 (M+H), (>95% purity). ¹H NMR was consistent withthe structure.

R═N(H)(CH₂)₃Ph, see Example 17.

Example 30 Preparation of Compound 15m

Using p-cyclohexenyl phenethylamine in the general procedure above gavethe title compound. LCMS m/z 801 (M+H), (>95% purity). ¹H NMR wasconsistent with the structure.

R=

see Example 17. Example 31 Preparation of Compound 15n

Using o-methoxyphenethylamine in the general procedure above gave thetitle compound. LCMS m/z 749 (M+H), (>95% purity). ¹H NMR was consistentwith the

R=

see Example 17. Example 32 Preparation of Compound 15o

Using p-fluorophenethylamine in the general procedure above gave thetitle compound. LCMS m/z 737 (M+H), (>95% purity). ¹H NMR was consistentwith the structure.

R=

see Example 17. Example 33 Preparation of Compound 15p

Using β-methylphenethylamine in the general procedure above gave thetitle compound. LCMS m/z 733 (M+H), (>95% purity). ¹H NMR was consistentwith the structure.

R═N(H)C(H)(CH₃)CH₂Ph, see Example 17.

Example 34 Preparation of Compound 15q

Using p-(trifluoromethyl)phenethylamine in the general procedure abovegave the title compound. LCMS m/z 787 (M+H), (>95% purity). ¹H NMR wasconsistent

with

R=

see Example 17. Example 35 Preparation of Compound 15r

Using p-methoxyphenethylamine in the general procedure above gave thetitle compound. LCMS m/z 749 (M+H), (>95% purity). ¹H NMR was consistentwith the structure.

R=

see Example 17. Example 36 Preparation of Compound 15s

Using indoline in the general procedure above gave the title compound.LCMS m/z 723 (M+H), (>95% purity). ¹HNMR was consistent with thestructure.

R=

see Example 17. Example 37 Preparation of Compound 15t

Using β-hydroxy-N-methylphenethylamine in the general procedure abovegave the title compound. LCMS m/z 749 (M+H), (>95% purity). ¹H NMR wasconsistent with the structure.

R═N(CH₃)C(H)(OH)(CH₂)Ph, see Example 17.

Example 38 Preparation of Compound 15u

Using m-(trifluoromethyl)phenethylamine in the general procedure abovegave the title compound. LCMS m/z 787 (M+H), (>95% purity). ¹H NMR wasconsistent with the structure.

R=

see Example 17. Example 39 Preparation of Compound 15v

Using m-methoxyphenethylamine in the general procedure above gave thetitle compound. LCMS m/z 749 (M+H), (>95% purity). ¹H NMR was consistentwith the structure.

R=

see Example 17. Example 40 Preparation of Compound 15w

Using tryptamine in the general procedure above gave the title compound.LCMS m/z 766 (M+H), (>95% purity). ¹H NMR was consistent with thestructure.

R=

see Example 17. Example 41 Preparation of Compound 15x

Using 1-napthylethylamine in the general procedure above gave the titlecompound. LCMS m/z 773 (M+H), (>95% purity). ¹H NMR was consistent withthe

R=

see Example 17. Example 42 Preparation of Compound 15y

Using 4-(aminoethyl)pyridine in the general procedure above gave thetitle compound. LCMS m/z 726 (M+H), (>95% purity). ¹H NMR was consistentwith the structure.

R=

see Example 17. Example 43 Preparation of Compound 15z

Using 3-(aminoethyl)pyridine in the general procedure above gave thetitle compound. LCMS m/z 726 (M+H), (>95% purity). ¹H NMR was consistentwith the structure.

R=

see Example 17. Example 44 Preparation of Compound 15aa

Using 2-(aminoethyl)pyridine in the general procedure above gave thetitle compound. LCMS m/z 726 (M+H), (>95% purity). ¹H NMR was consistentwith the

see Example 17.

Example 45 Synthesis of Compound 16 Synthesis of Compound 16a

To a stirred solution of Compound 2 (1.35 g, 0.98 mmol) in drydichloromethane (20 mL) was added 2,4,6-collidine (1.07 g, 8.82 mmol)and TBDMSOTf (1.811 g, 6.86 mmol) at 0° C. The reaction mixture wasslowly brought to room temperature and stirred for 12 hours. A few dropsof water was added to quench the excess TBSOTf, followed by extractionwith dichloromethane. The organic layer was washed with brine and driedover anhydrous Na₂SO₄, followed by concentration of the solvent to givethe corresponding crude product. The crude product was purified by flashcolumn chromatography to give Compound 16a (1.048 g, 55%).

[α]_(D)=+16° (c 0.6, CHCl₃). ESI/MS calcd for C₁₀₀H₁₄₉N₅O₂₄Si₅ (M+H⁺)1944.94; found 1946.

Synthesis of Compound 16b

To a stirred solution of Compound 16a (330 mg, 0.17 mmol) in dry DMF (6mL) was added 60% NaH in mineral oil (8 mg) at 0° C. with stirringcontinued for an additional 6 hours at 0° C. A few drops of saturatedammonium chloride solution were added, followed by extraction with ethylacetate. The organic layer was washed with brine and dried overanhydrous Na₂SO₄, followed by concentration of the solvent yielded thecorresponding crude product. The crude product was purified by flashcolumn chromatography to yield the Compound 16b (180 mg, 58%) and 120 mg(36%) of Compound 16a was also recovered.

[α]_(D)=+18° (c 0.5, CHCl₃). ESI/MS calcd for C₉₃H₁₄₁N₅O₂₃Si₅ (M+H⁺)1836.89; found 1837.6

Synthesis of4′,6′-O-benzylidene-penta-O-tert-butyldimethylsilanyloxy-tetra-N-benzyloxycarbonylparomomycin (16c)

To a stirred solution of Compound 16b (190 mg, 0.1 mmol) in DMF (7 mL)was added 0.7 mL of aqueous LiOH (9 mg, 0.21 mmol) with stirringcontinued for an additional 3 hours at room temperature. A few drops ofsaturated ammonium chloride solution was added, followed by extractionwith ethyl acetate. The organic layer was washed with brine and driedover anhydrous Na₂SO₄, followed by concentration of the solvent yieldedthe corresponding crude product. The crude product was purified by flashcolumn chromatography to Compound 16c (100 mg, 53%) and 50 mg (26%) ofCompound 16b was also recovered.

[α]_(D)=+13° (c 0.3, CHCl₃), ESI/MS calcd for C₉₂H₁₄₃N₅O₂₂Si₅ (M+H⁺)1810.91; found 1811.3.

Synthesis of4′,6′-O-Benzylidene-penta-O-tert-butyldimethylsilanyloxy-tetra-N-benzyloxycarbonyl-N-1-habaparomomycin (16d)

To a stirred solution of benzyloxy 4-hydroxy aminobutric acid (27 mg,0.11 mmol), N-Hydroxy succinimide (12 mg, 0.11 mmol) in dry THF (2 mL)was added DCC (22 mg, 0.11 mmol) with stirring continued for anadditional 1 hour at room temperature. To this reaction mixture the freeamine, Compound 16c (95 mg, 0.053 mmol) in dry THF (2 mL) and triethylamine (15 μL, 0.11 mmol) was added with stirring for 12 hours at roomtemperature. Evaporation of the solvent followed by purification byflash column chromatography gave Compound 16d (80 mg, 74%).

[α]_(D)=+19° (c 0.4, CHCl₃).

Synthesis of 4′,6′-O-benzylidene-tetra-N-benzyloxycarbonyl-N-1-habaparomomycin (16e)

Compound 16d (90 mg, 0.044 mmol) was dissolved in dry pyridine (2 mL),HF.Py (2 mL) was added at 0° C., the reaction was slowly brought to roomtemperature and stirred for 2 days. Water was added and the reactionmixture was extracted with ethyl acetate followed by washing with brine.The organic layer was dried over Na₂SO₄ and evaporated to give the crudeproduct. The crude product was purified by column chromatography to giveCompound 16e (50 mg, 77%).

[α]_(D)=+20° (c 0.6, CHCl₃). ESI/MS calcd for C₇₄H₈₆N₆O₂₆ (M+H⁺);1475.56; found 1475.7.

Synthesis of Compound 16

To a solution of Compound 16e (270 mg, 0.183 mmol) in pyridine (2 mL)was added acetic anhydride (1 mL) with stirring maintained for 24 hoursat room temperature. Water (10 mL) was added and the precipitatedproduct was filtered. The aqueous layer was extracted with ethylacetate, washed with saturated CuSO₄, brine and the organic layer wasdried over anhydrous Na₂SO₄. The organic layer was combined with theprecipitated product and evaporated to provide the crude material, whichyielded Compound 16 (300 mg, 93%) after column chromatography.

[α]_(D)=+7.5° (c 0.2, CHCl₃). ESI/MS calcd for (M+H⁺) 1768.63; found1769.8.

Example 46 Synthesis of Compound 17

Compound 16 (300 mg, 0.17 mmol) was stirred in 20 mL of aceticacid/water mixture (4:1) at room temperature for 4 days. Water was addedand the precipitated product was filtered. The aqueous layer wasextracted with ethyl acetate, washed with water, brine and the organiclayer was dried over anhydrous Na₂SO₄. The organic layer was combinedwith the precipitated product and evaporated to yield the crudematerial, which yielded Compound 17 (280 mg, 98%) after columnchromatography.

[α]_(D)=+10.7° (c 0.3, CHCl₃). HRMS calcd for (M+H⁺) 1681.60911; found1681.60830.

Example 47 Synthesis of Compound 18

To a solution of Compound 17 (290 mg, 0.17 mmol) in pyridine (2 mL) wasadded TsCl (36 mg, 0.19 mmol), DMAP (5 mg, 0.041 mmol) with stirringmaintained for 12 hours at room temperature. An additional 1.1equivalent of TsCl (36 mg, 0.19 mmol) was added and the reaction wasstirred for additional 8 hours at room temperature. Water was added andthe precipitated product was filtered. The aqueous layer was extractedwith ethyl acetate, washed with water, brine and the organic layer wasdried over anhydrous Na₂SO₄. The organic layer was combined with theprecipitated product and evaporated to yield the crude material.Compound 18 (300 mg, 96%) was obtained after column chromatography.

[α]_(D)=+14.8° (c 0.25, CHCl₃). HRMS calcd for C₈₈H₁₀₂N₆O₃₅S (M+H⁺)1835.61796; found 1835.61976.

Example 48 Synthesis of Compound 19

To a solution of Compound 18 (320 mg, 0.175 mmol) in dry DMF (3 mL) wasadded NaN₃ (113 mg, 1.74 mmol) with stirring maintained for 24 hours 70°C. Water was added and the resulting mixture was extracted with ethylacetate followed by washing with water and then brine. The organic layerwas dried over anhydrous Na₂SO₄ and evaporated under reduced pressure.Compound 19 (252 mg, 84%) was obtained following column chromatography.

[α]_(D)=+11.3° (c 0.3, CHCl₃). ESI/MS calcd for C₈₁H₉₅N₉O₃₂ (M+H⁺)1705.61; found 1707.0.

Example 49 Synthesis of Compound 20

Very small piece of sodium was added into methanol (10 mL) and pH wasadjusted to 10. This solution was transferred to Compound 19 in methanol(1 mL) and stirred overnight (12 hours). Dry ice was added to quench thereaction followed by evaporation of the methanol. The resultant crudematerial was purified by column chromatography to yield Compound 20 (52mg, 66%).

[α]_(C)=+16° (c 0.15, CHCl₃). HRMS calcd for C₆₇H₈₁N₉O₂₅ (M+H⁺)1412.54164; found 1412.53764.

Example 50 Synthesis of Compound 21

To a solution of Compound 20 (30 mg, 0.021 mmol) in dry THF (3 mL) wasadded 1M PMe₃ in THF (26 μL, 0.026 mmol) with stirring maintained for 1hour at room temperature. Water (0.2 mL) was added and stirring wascontinued for an additional hour. Another 26 μL of PMe₃ (1 M in THF) wasadded and stirred for 12 hours. Evaporation of the reaction mixturefollowed dissolving in ethyl acetate and washing with water gave thecrude product which was pure enough to use in the next step. To thiscrude amine in dry methanol was added phenylproyl aldehyde (3 mg, 0.022mmol) and a drop of glacial acetic acid with stirring for 5 minutesfollowed by addition of 1M NaBH₃CN in THF (42 μL, 0.042 mmol) withstirring for 12 hours at room temperature. Evaporation of the solventfollowed by column purification gave Compound 21 (12 mg, 38%, 2 steps).

[α]_(C)=+16.7° (c 0.15, CHCl₃). ESI/MS calcd for C₇₆H₉₃N₇O₂₅ (M+H⁺)1503.62; found 1504.7.

Example 51 Synthesis of Compound 22 (N-1-haba-6′-phenylpropyl neomycin)

To a solution of Compound 21 (11 mg, 0.0073 mmol) in 2 mL of aceticacid/water mixture (4:1) and 0.5 mL of methanol was added 20% Pd(OH)₂(22 mg) at room temperature with stirring for 6 hours under anatmosphere of hydrogen(balloon). The material was filtered over celiteand lypholized to give Compound 22 as the acetic acid salt (8 mg, 99%).

[α]_(D)=+39.2° (c 0.12, H₂O). NMR (400 MHz, D₂O) δ 7.28-7.17 (m, 5H),5.89 (d, J=3.7 Hz, 1H), 5.29 (s, 1H), 5.16 (s, 1H), 4.45-4.37 (m, 1H),4.32-4.25 (m, 1H), 4.25-4.15 (m, 2H), 4.1 (br s, 2H), 3.95-3.72 (m, 6H),3.7-3.45 (m, 4H), 3.4-3.2 (m, 6H), 3.19-3.1 (m, 1H), 3.01-2.94 (m, 4H),2.6 (t, J=7.4 Hz, 2H), 2.15-1.8 (m, 6H). ¹³C NMR (125 MHz, D₂O) δ 175.2,140.2, 128.4, 128.0, 126.1, 109.9, 95.1, 94.9, 85.3, 80.8, 75.4, 74.6,73.3, 73.2, 70.5, 69.6, 69.1, 68.8, 67.5, 67.2, 66.9, 59.4, 53.0, 50.4,48.4, 48.2, 47.8, 47.7, 40.0, 36.2, 31.4, 30.4, 29.5, 26.5. HRMS calcdfor C₃₆H₆₃N₇O₁₅ (M+11) 834.44549; found 834.44463.

Example 52 Synthesis of Compound 23

To a solution of Compound 20 (18 mg, 0.0127 mmol) in 2 mL of aceticacid/water mixture (4:1) and 0.2 mL of methanol was added 20% Pd(OH)₂(18 mg) at room temperature with stirring maintained for 2 hours underan atmosphere of hydrogen (balloon). The material was filtered overcelite and lypholized to give Compound 23 as the acetic acid salt (13mg, 95%).

[α]_(D)=+27.4° (c 0.23, H₂O). ¹H NMR (400 MHz, D₂O) δ 5.89 (s, 1H), 5.28(s, 1H), 5.15 (s, 1H), 4.38 (br s, 1H), 4.27 (br s, 1H), 4.17 (br s,2H), 4.1 (br s, 2H), 3.9-3.75 (m, 6H), 3.69-3.62 (m, 2H), 3.5-3.4 (m,2H), 3.4-3.2 (m, 6H), 3.8-3.1 (m, 1H), 3.0 (br s, 2H), 2.18-1.98 (m,2H), 1.7-1.6 (m, 2H). ¹³C NMR (125 MHz, D₂O) δ 175.2, 109.9, 95.1, 94.9,85.2, 80.8, 75.5, 74.6, 73.3, 73.1, 70.3, 69.7, 69.0, 68.9, 67.6, 67.2,66.9, 59.4, 53.1, 50.4, 48.4, 48.2, 40.0, 39.7, 36.2, 30.4, 29.5. HRMScalcd for C₂₇H₅₃N₇O₁₅ (M+H⁺) 716.367; 24 found 716.36662.

Example 53 Synthesis ofN-1-aloc-4′,6′-O-benzylidene-penta-O-tert-butyldimethylsilanyloxy-penta-N-benzyloxycarbonylparomomycin (25)

To a stirred solution of Compound 16c (Example 45, 1.125 g, 0.62 mmol)in dry dichloromethane (20 mL) was added Et₃N (0.11 mL, 1.24 mmol) andalloc-Cl (83 μL, 0.78 mmol) at 0° C. The reaction mixture was slowlybrought to room temperature and stirred for 6 hours. Evaporation of thesolvent followed by purification by flash column chromatography yieldedCompound 25 (600 mg, 51%).

[α]_(D)=+5.25° (c 0.4, CHCl₃). ESI/MS calcd for C₉₆H₁₄₇N₅O₂₄Si₅ (M+H⁺)1894.93; found 1895.3.

Example 54 Synthesis ofN-1-aloc-6-O-allyl-4′,6′-O-benzylidene-penta-O-tert-butyldimethylsilanyloxy-penta-N-benzyloxycarbonylparomomycin (26)

To a stirred solution of Compound 25 (558 mg, 0.3 mmol) in dry THF (15mL) were added 0.5M KHMDS in toluene (0.66 mL, 0.33 mmol) and allyliodide (0.11 mL, 1.2 mmol) at 0° C. The reaction mixture was slowlybrought to room temperature and stirred for 12 hours. The reactionmixture was quenched with saturated NH₄Cl solution followed byextraction with ethyl acetate. The organic layer was washed with brineand dried over anhydrous Na₂SO₄ and evaporated under reduced pressure togive the crude product. The crude material was purified by flash columnchromatography to yield Compound 26 (480 mg, 83%).

[α]_(D)=+10.1 (c 0.6, CHCl₃).

Example 55 Synthesis of6-O-allyl-4′,6′-O-benzylidene-penta-O-tert-butyldimethylsilanyloxy-N-1-haba-penta-N-benzyloxycarbonylparomomycin (27)

To a solution of Compound 26 (1.125 g, 0.62 mmol) and morpholine in dryTHF (20 mL) was added Pd(PPh₃)₄ (29 mg, 0.025 mmol) at room temperaturewith stirring for 3 hours. Evaporation of the solvent yielded the crudefree amine and this was used in the next step without purification.

To a solution of benzyloxy 4-hydroxy aminobutric acid (253 mg, 1 mmol),N-hydroxy succinimide (115 mg, 1 mmol) in dry THF (2 mL) was added DCC(201 mg, 1 mmol) with stirring maintained for 2 hours at roomtemperature. To this reaction mixture the crude free amine (from above)in dry THF (2 mL) and triethyl amine (0.11 mL, 0.76 mmol) was added withstirring for 12 hours at room temperature. Evaporation of the solventfollowed by purification by flash column chromatography yielded Compound27 (160 mg, 31%).

[α]_(D)=+11.0° (c 0.1, CHCl₃).

Example 56 Synthesis of4′,6′O-benzylidene-N-1-haba-6-O-phenylethylaminoethyl-penta-N-benzloxycarbonylparomomycin (28a)

Ozone gas was passed through a stirred solution of Compound 27 (78 mg,0.037 mmol) in dry dichloromethane (3 mL) at −78° C. for 2 hours. Ozonewas degassed by passing nitrogen gas for 10 minutes followed by theaddition of excess dimethyl sulfide (0.2 mL). This solution was stirredfor 2 hours at room temperature. The solvent was reduced under reducedpressure and the remaining mixture was extracted with ethyl acetate. Theorganic layer was washed with NaHCO₃, brine and dried over anhydrousNa₂SO₄. Evaporation of the solvent gave the crude material (75 mg). Thismaterial was dissolved in McOH. To this reaction mixture phenylethylamine (10 mg, 0.083 mmol) and one drop of AcOH were added and stirredfor 5 minutes. Then NaBH₃CN (5 mg, 0.08 μmol) was added and stirred for12 hours at room temperature. Evaporation of the solvent followed gavethe crude product (58 mg). This material was dissolved in dry pyridine(1 mL) followed by the addition of HF.Py (1 mL) at 0° C. The reactionwas slowly brought to room temperature and stirred for 2 days. Water wasadded to the reaction mixture followed by extraction with ethyl acetate.The organic layer was washed with brine and dried over Na₂SO₄.Evaporation of the solvent gave the crude material and this crudeproduct was purified by column chromatography to give Compound 28a (23mg, 38%, 3 steps).

[α]_(D)=+15.8° (c 0.3, CHCl₃). ESI/MS calcd for C₈₄H₉₉N₇O₂₆ (M+H⁺)1622.66; found 1623.1.

Example 57 Synthesis of N-1-haba-6-O-phenylethylaminoethyl-paromomycin(29a)

Compound 28a (22 mg, 0.014 mmol) in 2 mL of acetic acid/water mixture(4:1) was stirred at room temperature for 12 hours and then for anadditional 6 hours at 55° C. To this reaction mixture 20% Pd(OH)₂ (22mg) was added and stirred under an atmosphere of hydrogen (balloon) for3 hours. The mixture was filtered over celite and lypholized to give thepure acetate salt of Compound 29a (14 mg, 81%).

[α]_(D)=+40.33° (c 0.25, H₂O). ¹H NMR (400 MHz, D₂O) δ 7.29-7.20 (m,5H), 5.65 (d, J=3.8 Hz, 1H), 5.24 (s, 1H), 5.14 (s, 1H), 4.39-4.37 (m,1H), 4.5-4.23 (m, 3H), 4.1-4.0 (m, 2H), 3.9-3.71 (m, 6H), 3.65-3.58 (m,5H), 3.56-3.23 (m, 12H), 3.01-2.97 (m, 2H), 2.92-2.9 (m, 2H), 2.11-2.0(m, 2H), 1.68-1.6 (m, 2H). ¹³C NMR (125 MHz, D₂O) δ 176.4, 136.8, 129.9,129.6, 128.3, 110.9, 96.8, 96.1, 86.0, 81.9, 78.4, 75.9, 74.5, 74.2,71.0, 70.3, 70.0, 69.6, 68.3, 67.9, 61.0, 60.8, 54.6, 51.6, 50.0, 49.9,49.7, 49.3, 44.7, 44.1, 37.9, 32.5, 31.6, 30.7. ESI/MS calcd forC₃₇H₆₅N₇O₁₆ (M+H⁺) 864.45; found 864.8.

Example 58 Synthesis of4′,6′-O-benzylidene-N-1-haba-6-O-(1,3-diaminoethyl)-penta-N-benzyloxycarbonylparomomycin (28b)

Ozone gas was passed to a stirred solution of Compound 27 (78 mg, 0.037mmol) in dry dichloromethane (3 mL) at −78° C. for 2 hours. Ozone wasdegassed by passing nitrogen gas for 10 minutes. To this solution wasadded excess dimethyl sulfide (0.2 mL). with stirring for 2 hours atroom temperature. Then the solvent was evaporated under reduced pressureand the material was extracted with ethyl acetate. The organic layer waswashed with NaHCO₃ and brine then dried over anhydrous Na₂SO₄.Evaporation of the solvent gave the crude material (75 mg). Thismaterial was dissolved in MeOH and NH-Cbz-(CH₂)₂CH₂NH₂ (15 mg, 0.072mmol) and one drop of AcOH were added with stirring for 5 minutes.NaBH₃CN (5 mg, 0.081 mmol) was added with stirring for 12 hours at roomtemperature. Evaporation of the solvent followed by usual work up gavethe crude product (58 mg). This material was dissolved in dry pyridine(1 mL) followed by the addition of HF.Py (1 mL) at 0° C. and thereaction was slowly brought to room temperature and stirred for 2 days.Water was added to the reaction mixture and extracted with ethyl acetatefollowed by washing with brine and the organic layer was dried overNa₂SO₄. Evaporation of the solvent gave the crude material and thiscrude product was purified by column chromatography to give Compound 28b(20 mg, 31%, 3 steps).

[α]_(D)=+15.2° (c 0.4, CHCl₃). ESI/MS calcd for C₈₇H₁₀₄N₈O₂₈ (M+H⁺)1709.7; found 1710.4.

Example 59 Synthesis of N-1-haba-6-O-(1,3-diaminoethyl) paromomycin(29b)

Compound 28b (20 mg, 0.012 mmol) in 2 mL of acetic acid/water mixture(4:1) was stirred at room temperature for 12 hours followed by anadditional 6 hours at 55° C. To this reaction mixture 20% Pd(OH)₂ (20mg) was added under an atmosphere of hydrogen (balloon) for 3 hours. Thematerial was filtered over celite and lypholized to give the acetatesalt of Compound 29b (13 mg, 87%).

[α]_(D)=+27.33° (c 0.5, H₂O). ¹H NMR (400 MHz, D₂O) δ 5.66 (s, 1H), 5.24(s, 1H), 5.15 (s, 1H), 4.38-4.37 (m, 1H), 4.29-4.08 (m, 6H), 3.84-3.75(m, 8H), 3.67-3.64 (m, 5H), 3.5-3.26 (m, 7H), 3.19-2.93 (m, 6H),2.08-1.94 (m, 4H), 1.68-1.59 (m, 2H). ¹³C NMR (125 MHz, D₂O) δ 176.4,110.8, 96.8, 96.3, 85.9, 82.0, 78.5, 76.2, 74.5, 74.4, 74.3, 71.0, 70.3,70.0, 69.6, 68.3, 68.0, 61.0, 60.9, 54.6, 51.6, 50.1, 49.8, 49.3, 45.8,44.8, 44.2, 37.4, 37.2, 31.7, 30.7, 24.7. ESI/MS calcd for C₃₂H₆₄N₈O₁₆(M+H⁺) 817.44; found 817.8.

Example 60

General Procedure for the Synthesis of Aminoglycoside Compounds withRing IV Removed (30a-c)

Ring IV was eliminated from the common intermediate, Compound 4, usinglead tetraacetate (see Hanessian, S.; Takamoto, T. J. Antibiotics, 1974,46, 4009-4012 and Hanessian S.; Takamoto T.; Masse R.; Patil G. Can. J.Chem. 1978, 56, 1482). Following the procedures illustrated in theprevious examples (protection, generation of an aldehyde, reductiveamination and deprotection) such as examples 1-8, a wide variety of2″-modified derivatives can then be prepared. In particular the2″-substituted derivatives as described in examples 4-8 can be prepared.Three of the derivatives (30a, 30b and 30c) were prepared and have assaydata in subsequent examples.

Compound# R¹ R² 30a H H 30b H (CH₂)₃NH₂ 30c H (CH₂)₂Ph

See Example 8 for additional R¹R² groups.

Example 615″,6′-O-bis-tert-butyldimethylsilanyloxy-penta-N-benzyloxycarbonyl-3′,4′-dideoxyparomomycin (32)

Compound 31 is prepared as per Battistini et al., Semisyntheticaminoglycoside antibiotics, IV, 3′,4′-Dideoxyparomomycin and analogs. J.Antibiotics 1982, 35, 98-101. Alternatively, Compound 31 is preparedaccording to the scheme of this example from Compound 17, then protectedwith Cbz groups as described in Example 1. To a solution of Compound 31(272 mg, 0.22 mmol) and imidazole (64 mg, 0.91 mmol) in drydichloromethane (5 mL) was added TBSC1 (77 mg, 0.51 mmol) at roomtemperature with stirring for 24 hours. A few drops of water were addedto quench the excess TBSC1 and the mixture was extracted with ethylacetate. The organic layer was washed with saturated brine and driedover anhydrous Na₂SO₄, followed by concentration of the solvent to givethe crude product. The crude product was purified by flash columnchromatography to yield Compound 32 (225 mg, 68%).

[α]_(D)=+29° (c 0.7, CHCl₃). HRMS calcd for C₇₅H₁₀₃N₅O₂₂Si₂ (M+H⁺):1482.67060; found: 1482.66832.

Example 622″-O-allyl-5″,6′-O-bis-tert-butyldimethylsilanyloxy-penta-N-benzyloxycarbonyl-3′,4′-dideoxyparomomycin (33)

To a stirred solution of Compound 32 (222 mg, 0.15 mmol) and allyliodide (70 μL, 0.75 mmol) in dry THF (5 mL) was added 0.5 M KHMDS in THF(300 μL, 0.15 mmol) at 0° C. The reaction mixture was slowly brought toroom temperature and stirred for 12 hours. A few drops of saturatedNH₄Cl solution were added to quench the reaction and the mixture wasextracted with ethyl acetate. The organic layer was washed withsaturated brine, dried over anhydrous Na₂SO₄ and concentrated to givethe corresponding crude product. The crude product was purified by flashcolumn chromatography to give Compound 33 (155 mg, 68%).

[α]_(D)=+18.75° (c 0.4, CHCl₃). ESI/MS calcd for C₇₈H₁₀₇N₅O₂₂Si₂ (M+H⁺):1522.69; found: 1522.7.

Example 632″-O-allyl-tetra-tert-butyldimethylsilanyloxy-penta-N-benzyloxycarbonyl-3′,4′-dideoxyparomomycin (34)

To a stirred solution of Compound 33 (800 mg, 0.53 mmol) in drydichloro-methane (15 mL) was added 2,4,6-collidine (321 mg, 2.65 mmol)and TBSOTf (693 mg, 2.65 mmol) at 0° C. The reaction mixture was slowlybrought to room temperature and stirred for 12 hours. A few drops ofwater were added to quench the excess TBSOTf, followed by extractionwith dichloromethane. The organic layer was washed with saturated brine,dried over anhydrous Na₂SO₄ and concentrated to give the crude product.The crude product was purified by flash column chromatography to yieldCompound 34 (715 mg, 77%).

[α]_(D)=+10.33° (c 0.6, CHCl₃). ESI/MS calcd for C₉₀H₁₃₅N₅O₂₂Si₄ (M+H⁺):1750.87; found: 1751.4.

Example 64 Cyclic Carbamate (35)

To a solution of Compound 34 (692 mg, 0.4 mmol) in dry DMF (10 mL) wasadded 60% NaH in mineral oil (19 mg) at 0° C. with stirring continued 6hours at 0° C. A few drops of saturated ammonium chloride solution wereadded, followed by extraction with ethyl acetate. The organic layer waswashed with saturated brine, dried over anhydrous Na₂SO₄ andconcentrated to give the crude product. The crude product was purifiedby flash column chromatography to yield Compound 35 (323 mg, 49%) and180 mg (26%) of the starting material Compound 34 was also recovered.

[α]_(D)=+18.33° (c 0.3, CHCl₃). ESI/MS calcd for C₈₃H₁₂₇N₅O₂₁Si₄ (M+H⁺):1642.81; found: 1643.5.

Example 652″-O-allyl-tetra-tert-butyldimethylsilanyloxy-tetra-N-benzyloxycarbonyl-3′,4′-dideoxyparomomycin (36)

To a solution of Compound 35 (350 mg, 0.21 mmol) in DMF (5 mL) was added0.5 mL of aqueous LiOH (18 mg, 0.43 mmol) with stirring continued for 4hours at room temperature. A few drops of saturated ammonium chloridesolution were added, followed by extraction with ethyl acetate. Theorganic layer was washed with saturated brine, dried over anhydrousNa₂SO₄ and concentrated to give the crude product. The crude product waspurified by flash column chromatography to give Compound 36 (300 mg,88%).

[α]_(D)=+20.33° (c 0.5, CHCl₃). ESI/MS calcd for C₈₂H₁₂₉N₅O₂₀Si₄(M+H⁺):1616.83; found: 1617.4.

Example 662″-O-allyl-tetra-tert-butyldimethylsilanyloxy-penta-N-benzyloxycarbonyl-3′,4′-dideoxy-N-1-habaparomomycin (37)

To a solution of benzyloxy 4-hydroxy aminobutric acid (66 mg, 0.26mmol), N-hydroxy succinimide (121 mg, 1.05 mmol) in dry THF (10 mL) wasadded DCC (216 mg, 1.05 mmol) with stirring continued for 1 hour at roomtemperature. To this reaction mixture the free amine, Compound 36 (340mg, 0.21 mmol) in dry THF (2 mL) and triethyl amine (0.2 mL, 0.42 mmol)were added with stirring for 12 hours at room temperature. Evaporationof the solvent and purification of the residue by flash columnchromatography gave Compound 37 (290 mg, 75%).

[α]_(D)=+16.67° (c 0.12, CHCl₃). ESUMS calcd for C₉₄H₁₄₂N₆O₂₄Si₄ (M+H⁺):1851.91; found: 1852.8.

Example 672″-O-phenylethylaminoethyl-penta-N-benzyloxycarbonyl-3′,4′-dideoxy-N-1-habaparomomycin (38)

Ozone gas was passed through a stirred solution Compound 37 (135 mg,0.073 mmol) in dry dichloromethane (5 mL) at −78° C. for 2 hours. Thenthe excess ozone was degassed by passing nitrogen gas for 10 minutesfollowed by the addition of excess dimethyl sulfide (0.1 mL). Thissolution was stirred for 2 hours at room temperature. The solvent wasremoved under reduced pressure and the resulting material was extractedwith ethyl acetate, washed with NaHCO₃ and brine and the organic layerwas dried over anhydrous Na₂SO₄. Evaporation of the solvent gave thecrude material (125 mg). This material was dissolved in MeOH and phenylethyl amine (16 mg, 0.13 mmol) and one drop of AcOH was added withstirring for 5 minutes. NaBH₃CN (9 mg, 0.15 mmol) was added and stirredfor 12 hours at room temperature. Evaporation of the solvent gave thecrude product (70 mg). This material was dissolved in dry pyridine (1mL) followed by the addition of HF.Py (1 mL) at 0° C. and the reactionwas slowly brought to room temperature and stirred for 2 days. Water wasadded and the mixture was extracted with ethyl acetate, washed withbrine and the resulting organic layer was dried over Na₂SO₄. Evaporationof the solvent gave the crude material which was further purified bysilica gel flash column chromatography to give Compound 38 (33 mg, 30%).

[α]_(D)=+20.7° (c 0.2, CHCl₃). ESI/MS calcd for C₇₇H₉₅N₇O₂₄(M+H⁺):1502.64; found: 1504.1.

Example 68 2″-O-phenylethylaminoethyl-3′,4′-dideoxy-N-1-haba paromomycin(39)

To a stirred solution of Compound 38 (20 mg, 0.013 mmol) in AcOH/water(4:1) mixture was added 20% Pd(OH)₂ (20 mg) and stirred under anatmosphere of hydrogen using a hydrogen balloon for 2 hours. Filterationover celite followed by lypholyzation gave Compound 39 (16 mg,quantitative).

[α]_(D)=+33.33° (c 0.15, H₂O). ¹H NMR (400 MHz, D₂O) δ 7.4-7.1 (m, 5H),5.5 (s, 1H), 5.28 (s, 1H), 5.08 (s, 1H), 4.5-4.4 (m, 1H), 4.2-4.0 (m,5H), 3.9-3.6 (m, 9H), 3.5-3.1 (m, 12H), 3.0-2.8 (m, 4H), 2.1-1.4 (m,8H); ¹³C NMR (125 MHz, D₂O) δ 175.9, 136.6, 129.4, 129.1, 127.7, 108.6,95.5, 95.0, 86.0, 81.0, 80.8, 77.5, 74.0, 73.8, 71.1, 70.7, 70.0, 68.0,67.6, 65.6, 63.6, 59.9, 51.1, 49.7, 49.2, 49.1, 48.8, 47.2, 40.6, 37.0,31.9, 31.2, 30.32, 24.25, 21.36; ESI/MS calcd for C₃₇H₆₅N₇O₁₄ (M+H⁺):832.45895; found: 832.46627.

Example 692″-O-allyl-4′,6′-O-benzylidene-tetra-O-tert-butyldimethylsilanyloxy-penta-N-benzyloxycarbonylparomomycin (40)

To a stirred solution of Compound 4a (3.8 g, 2.49 mmol) in drydichloromethane (80 mL) was added 2,4,6-collidine (1.8 g, 1.97 mmol) andTBSOTf (3.94 g, 14.92 mmol) at 0° C. The reaction mixture was slowlybrought to room temperature and stirred for 12 hours. A few drops ofwater were added to quench the excess TBSOTf and the mixture wasextracted with dichloromethane. The organic layer was washed withsaturated brine, dried over anhydrous Na₂SO₄ and the solvent was removedunder reduced pressure to give the crude product. The crude product waspurified by flash column chromatography to give Compound 40 (1.6 g, 35%)and 1 g of (20%) the corresponding fully TBS protected compound.

[α]_(D)=+11.0° (c 1, CHCl₃). ESI/MS calcd for C₉₇H₁₃₉N₅O₂₄Si₄ (M+H⁺):1870.89; found: 1871.6.

Example 70 Synthesis of the Cyclic Carbamate (41)

To a solution of Compound 40 (1.47 g, 0.783 mmol) in dry DMF (20 mL) wasadded 60% NaH in mineral oil (36 mg) at 0° C. with stirring continuedfor an additional 6 hours at 0° C. A few drops of saturated ammoniumchloride solution were and the mixture was extracted with ethyl acetate.The organic layer was washed with saturated brine, dried over anhydrousNa₂SO₄ and concentrated under reduced pressure to give crude product.The crude product was purified by flash column chromatography to giveCompound 41 (650 mg, 47%) and 560 mg (38%) of starting material Compound40 was also recovered. [α]_(D)=+20° (c 1, CHCl₃). ESI/MS calcd forC₉₀H₁₃₁N₅O₂₃Si₄ (M+H⁺): 1762.83; found: 1763.2.

Example 712″-O-allyl-4′,6′-O-benzylidene-tetra-O-tert-butyldimethylsilanyloxy-tetra-N-benzyloxycarbonylparomomycin (42)

To a solution of Compound 41 (730 mg, 0.41 mmol) in DMF (10 mL) wasadded 1 mL of aqueous LiOH (35 mg, 0.83 mmol) with stirring continuedfor an additional 6 hours at room temperature. A few drops of saturatedammonium chloride solution were added and the mixture was extracted withethyl acetate. The organic layer was washed with saturated brine, driedover anhydrous Na₂SO₄ and concentrated to give crude product. The crudeproduct was purified by flash column chromatography to give Compound 42(450 mg, 63%) and Compound 41 (264 mg, 36%) was also recovered.

[α]_(D)=+3.77° (c 0.45, CHCl₃). ESI/MS calcd for C₈₉H₁₃₃N₅O₂₂Si₄ (M+H⁺):1736.85; found 1737.2.

Example 722″-O-allyl-4′,6′-O-benzylidene-tetra-O-tert-butyldimethylsilanyloxy-penta-N-benzyloxycarbonyl-N-1-habaparomomycin (43)

To a solution of benzyloxy 4-hydroxy aminobutric acid (364 mg, 1.45mmol) and N-hydroxy succinimide (167 mg, 1.45 mmol) in dry THF (10 mL)was added DCC (299 mg, 1.45 mmol) with stirring continued for additional2 hours at room temperature. To this reaction mixture Compound 42 (500mg, 0.29 mmol) in dry THF (2 mL) and triethyl amine (0.2 mL, 1.45 mmol)was added with stirring for 12 hours at room temperature. Evaporation ofthe solvent followed by purification by flash column chromatography gaveCompound 43 (400 mg, 70%).

[α]_(D)=+13.7° (c 0.5, CHCl₃). ESI/MS calcd for C₁₀₁H₁₄₆N₆O₂₆Si₄ (M+H⁺):1971.94; found: 1972.7.

Example 73 Synthesis of Aldehyde (44)

Ozone gas was passed through a stirred solution of Compound 43 (400 mg,0.2 mmol) in dry dichloromethane (10 mL) at −78° C. for 2 hours. Theexcess ozone was degassed by passing nitrogen gas for 10 minutesfollowed by the addition of excess PPh₃ (210 mg, 0.8 mmol). Thissolution was stirred for 2 hours at room temperature. The solvent wasremoved under reduced pressure and the resulting material was extractedwith ethyl acetate, washed with NaHCO₃ and brine and the organic layerwas dried over anhydrous Na₂SO₄. Evaporation of the solvent gave thecrude material, which was purified by column chromatography to giveCompound 44 (270 mg, 67%).

[α]_(D)=+20.7° (c 0.9, CHCl₃). ESI/MS calcd for C₁₀₀H₁₄₄N₆O₂₇Si₄ (M+H⁺):1973.92; found: 1974.4.

Example 744′,6′-O-benzylidene-penta-N-benzyloxycarbonyl-N-1-haba-2″-O-phenylethylaminoethylparomomycin (45a)

To a stirred solution of Compound 44 (120 mg, 0.061 mmol) was addedphenyl ethyl amine (15 mg, 0.12 mmol) and one drop of AcOH with stirringfor 5 minutres. NaBH₃CN (8 mg, 0.12 mmol) was added with stirringmaintained for an additional 12 hours at room temperature. Evaporationof the solvent gave the crude product (120 mg). The crude product wasdissolved in dry pyridine (1 mL), HF.Py (1 mL) was added at 0° C. andthe reaction was slowly brought to room temperature and stirred for 2days. Water was added and the mixture was extracted with ethyl acetate.The organic phase was washed with brine and dried over Na₂SO₄.Evaporation of the solvent gave the crude material which was purified bycolumn chromatography to give Compound 45a (60 mg, 61%).

[α]_(D)=+10.5° (c 0.2, CHCl₃). ESI/MS calcd for C₈₄H₉₉N₇O₂₆ (M+H⁺):1622.66; found: 1623.1.

Example 75 N-1-haba-2″-O-phenylethylaminoethyl paromomycin (46a)

Compound 45a (30 mg, 0.019 mmol) in 3 mL of acetic acid and watermixture (4:1) was stirred at room temperature for 12 hours and at 55° C.for an additional 6 hours. To this reaction mixture 20% Pd(OH)₂ (30 mg)was added under an atmosphere of hydrogen (balloon) for 3 hours. Thereaction mixture was filtered through celite and lypholized to giveCompound 46a (21 mg, 91%).

[α]_(D)=+48.5° (c 0.2, H₂O). ¹H NMR (400 MHz, D₂O) δ 7.34-7.18 (m, 5H),5.7 (d, J=3.6 Hz, 1H), 5.33 (s, 1H), 5.1 (s, 1H), 4.5-4.49 (m, 1H),4.2-4.03 (m, 5H), 3.9-3.76 (m, 9H), 3.66-3.61 (m, 4H), 3.5-3.4 (m, 1H),3.38-3.18 (m, 10H), 3.0-2.97 (m, 2H), 2.92-2.89 (m, 2H), 2.1-2.0 (m,2H), 1.61-1.58 (m, 2H); ¹³C NMR (125 MHz, D₂O) δ 175.9, 136.6, 129.4,129.1, 127.8, 108.5, 96.3, 95.0, 86.0, 81.0, 80.7, 77.7, 74.0, 73.6,70.7, 69.8, 69.5, 69.2, 68.0, 67.6, 65.6, 60.5, 59.4, 54.2, 51.1, 49.5,49.1, 48.8, 47.3, 40.6, 37.0, 31.9, 31.2, 30.39, HRMS calcd forC₃₇H₆₅N₇O₁₆ (M+H⁺): 864.44878; found: 864.45613.

Example 764′,6′-O-benzylidene-penta-N-benzyloxycarbonyl-N-1-haba-2″-O-(1,3-diamino)ethylparomomycin (45b)

To a solution of Compound 44 (50 mg, 0.025 mmol) was addedN-Cbz-(CH₂)₂CH₂NH₂ (16 mg, 0.12 mmol) followed by one drop of AcOH withstirring for 5 minutes. NaBH₃CN (5 mg, 0.12 mmol) was added withstirring for 12 hours at room temperature. Evaporation of the solventgave the crude product. The crude product was dissolved in dry pyridine(1 mL) and HF.Py (1 mL) was added at 0° C. The reaction mixture wasslowly brought to room temperature and stirred for 2 days. Water wasadded and the mixture was extracted with ethyl acetate, washed withbrine and dried over Na₂SO₄. Evaporation of the solvent gave the crudematerial which was purified by column chromatography to give Compound45b (18 mg, 42%). [α]_(D)=+15.5° (c 0.4, CHCl₃). ESI/MS calcd forC₈₇H₁₀₄N₈O₂₈ (M+H⁺): 1709.70; found: 1710.0.

Example 77 N-1-haba-2″-O-(1,3-diamino)ethyl paromomycin (46b)

Compound 45b (18 mg, 0.011 mmol) in 1 mL of acetic acid and watermixture (4:1) was stirred for 12 hours at room temperature followed byan additional 6 hours at 55° C. 20% Pd(OH)₂ (18 mg) was added under anatmosphere of hydrogen (balloon) with stirring for 3 hours. The mixturewas filtered through celite and lypholized to give Compound 46b (14 mg,quantitative).

[α]_(D)=+30° (c 0.7, H₂O). ¹H NMR (400 MHz, D₂O) δ 5.7 (s, 1H), 5.4 (s,1H), 5.17 (s, 1H), 4.55-4.5 (m, 1H), 4.25-4.01 (m, 6H), 3.9-3.19 (m,20H), 3.06-2.9 (m, 6H), 2.1-1.55 (m, 6H); ¹³C NMR (125 MHz, D₂O) δ175.2, 107.7, 95.6, 94.2, 85.1, 81.3, 80.3, 79.8, 76.9, 73.3, 72.9,70.0, 69.1, 68.7, 68.4, 67.2, 66.8, 59.8, 58.7, 53.4, 50.4, 48.7, 48.3,46.9, 44.2, 42.0, 39.9, 36.3, 36.1, 30.4, 29.5, 23.1; HRMS calcd forC₃₂H₆₄N₈O₁₆ (M+H⁺): 817.44403; found: 817.45229.

Example 78 Synthesis of Orthogonally Protected Paromomycin (49)

A solution containing Compound 3 (540 mg, 0.362 mmol) andN,N-dimethylamino pyridine (176 mg, 1.44 mmol) in dry pyridine (20 mL)was treated with benzoyl chloride (0.85 mL, 7.25 mmol) at 0° C. Thereaction mixture was stirred at room temperature for 12 hours and at 70°C. for an additional 24 hours wherein the reaction was shown to hve goneto completion (tic) with the formation of two products with a 3:1 ratio.Water (1 mL) was added and after standing for 10 minutes, the solventwas removed under vacuum. The residue was dissolved in EtOAc/H₂O, theaqueous layer was extracted with EtOAc, and the combined organicextracts were washed with water, brine, dried over Na₂SO₄ andconcentrated under vacuum. The crude product was purified by silica gelflash column chromatography (2:3 EtOAc/hexane) to yield Compound 49 (510mg, 70%).

[α]_(D)+37.91° (c 1.15, CHCl₃); R_(f) 0.43 (1:1 EtOAc/hexane); FAB MScalcd for C₁₁₁H₁₁₃N₅O₂₉Si (M+H⁺) 2008.73, found 2008.7. The product witha 3′-OH free was isolated from column with 25% yield (173 mg);[α]_(D)+31.83° (c 1.2, CHCl₃); R_(f) 0.29 (1:1 EtOAc/hexane); FAB MScalcd for C₁₀₄H₁₀₉N₅O₂₇ (M+H⁺) 1904.64, found 1904.6.

Example 79 Selective Deblocking of the 5″-Position (50)

A solution of Compound 49 (420 mg, 0.209 mmol) in dry THF was treatedwith AcOH (119.6 uL, 2.09 mmol) and TBAF successively at 0° C. Thereaction mixture was allowed to come to room temperature and furtherstirred for 24 hours wherein the reaction had gone to completion. Thesolvent was removed under reduced pressure and the residue was dissolvedin EtOAc/H₂O, the aqueous layer was extracted with EtOAc, and thecombined organic extracts were washed with water, brine, dried overNa₂SO₄ and concentrated under vacuum. The crude product was purified bysilica gel flash chromatography (2:3 EtOAc/hexane) to yield Compound 50(202 mg, 51%).

[α]_(D)+25.16° (c 0.93, CHCl₃); R_(f) 0.47 (3:2 EtOAc/hexane); LCMScalcd for C₁₀₅H₉₉N₅O₂₉ (M+H⁺) 1894.93, found 1895.0. The product with5″-OH and one more additional OH free was isolated from column with 33%yield (125 mg); R_(f) 0.27 (3:2 EtOAc/hexane).

Example 80 Synthesis of 5″-O-Alkyl paromomycin analogues (51)

Compound 50 (120 mg, 0.063 mmol) was co-distilled with toluene twice anddissolved in dry THF (3 mL) in a flask covered with aluminum foil. Allyliodide (58.2 pt, 0.63 mmol) was added at 0° C. followed by the dropwiseaddition of 0.5 M KHMDS solution in toluene (152 μL, 0.076 mmol). Themixture mixture was stirred for 3 hours at room temperature by carefulmonitoring on TLC. The reaction mixture was quenched with an aqueoussolution of NH₄C1 (saturated, 0.1 mL) and the solvent was evaporated todryness in vacuo. The crude product was dissolved in EtOAc, washed withwater and the resultant product was purified by silica gel flashchromatography (1:2 EtOAc/hexane) to give the allyl ether (71 mg, 58%).

[α]_(D)+39.64° (c 0.84, CHCl₃); R_(f) 0.62 (1:1 EtOAc/hexane); LCMScalcd for C₁₀₈H₁₀₃N₅O₂₉ (M+H⁺) 1936.54, found 1936.6.

The allyl ether (100 mg, 0.0517 mmol) in CH₂Cl₂ (4 mL) was cooled at−78° C. and ozone was bubbled through for 2 hours after which argon wasbubbled through. The mixture was treated with PPh₃ (40.64 mg, 0.299mmol), warmed to the room temperature, solvent was removed under vacuumand the crude aldehyde was purified by silica gel flash chromatography(2:3 EtOAc/hexane) to give the aldehyde, Compound 51 (60 mg, 60%); R_(f)0.38 (1:1 EtOAc/hexane).

Example 81 Synthesis of 5″-O-Alkyl paromomycin analogues (52 and 53)

To a mixture Compound 51 (30 mg, 0.0155 mmol) and N,N-dimethylamine (2.0M in THF, 80 μL, 0.155 mmol) in dry MeOH (3 mL) was added AcOH (3-4drops) followed by NaBH₃CN (1.0 M in THF, 0.15 mL, 0.155 mmol). Themixture was stirred at room temperature overnight until disappearance ofCompound 51. The reaction mixture was diluted with EtOAc (10 mL) andwashed with a solution of NaHCO₃ (saturated, 2 mL) and dried overNa₂SO₄. After evaporation of the solvents, the residue was purified bysilica gel flash column chromatography (48:1 CH₂Cl₂/MeOH) to give thefully protected 5″-(2-dimethylamino) ethoxy derivative as white solid(26 mg, 85%).

[α]_(D)+21.97° (c 1.57, CHCl₃); R_(f) 0.67 (1:19 MeOH/CH₂Cl₂); LCMScalcd for C₁₀₉H₁₀₈N₆O₂₉ (M+H⁺) 1966.05, found 1966.4.

A solution of the fully protected 5″-(2-dimethylamino) ethoxy derivative(20 mg, 0.0102 mmol) in dry MeOH (2 mL) was treated with a catalyticamount of NaOMe in dry MeOH (1 mL, pH 8-9) and stirred at roomtemperature for 3 hours to completion of reaction. The reaction mixturewas neutralized by addition of dry-ice, and the solvent was evaporatedto dryness under vacuum. The crude product was purified by silica gelflash column chromatography (1:19 MeOH/CH₂Cl₂) to give Compound 52 (8.8mg, 60%).

[α]_(D)+18.54° (c 0.44, MeOH); R_(f) 0.32 (1:19 MeOH/CH₂Cl₂); LCMS calcdfor C₇₄H₈₈N₆O₂₄ (M+H⁺) 1445.59, found 1445.9.

The product with 6′″-N methylcarbamate, Compound 53 was isolated fromcolumn chromatography with 20% yield (3 mg).

[α]_(D)+15.6° (c 0.3, MeOH); R_(f) 0.32 (1:19 MeOH/CH₂Cl₂); LCMS calcdfor C₇₄H₈₈N₆O₂₄ (M+H⁺) 1368.23, found 1369.3.

Example 82 Synthesis of 5″-O-(2-N,N-dimethylamino ethyl) paromomycin(54), (55)

Compound 52 (6 mg, 0.0041 mmol) was dissolved in AcOH—H₂O (4:1, 2 mL)and heated at 60° C. for 2 hours to completion of reaction. The solventwas removed under reduced pressure and the crude product was dissolvedin MeOH—H₂O (1:1, 2 mL). 20% palladium hydroxide on carbon was added andthe suspension was stirred at room temperature overnight under anatmosphere of hydrogen (hydrogen balloon). The mixture was filteredthrough a layer of Celite, concentrated under vacuum, and the residuewas dissolved in AcOH—H₂O (2:1, 0.5 mL) and lyophilized to affordCompound 54 (4.1 mg, quantitative) as a white solid.

[α]_(D)+33.07° (c 0.26, H₂O); ¹H NMR (400 MHz, D₂O) δ 5.34 (s, 1H), 5.23(s, 11H), 5.0 (s, 1H), 4.33-4.12 (m, 4H), 4.11-4.0 (m, 1H), 3.94-3.83(m, 1H), 3.75-3.63 (m, 5H), 3.61-3.58 (m, 4H), 3.50-3.21 (m, 8H),3.13-2.93 (m, 3H), 2.78 (s, 6H), 2.17-2.08 (m, 1H), 1.82 (s, 15H),1.44-1.37 (m, 1H); LCMS calcd for C₂₇H₅₄N₆O₁₄) 687.37, found 687.6.

Compound 53 was also hydrogenolysed following the above procedure andlyophilized to give Compound 55 (2.3 mg, quantitative). ¹HNMR (400 MHz,D₂O) δ 5.36 (s, 1H), 5.25 (s, 1H), 5.0 (s, 1H), 4.35-4.14 (m, 4H),4.10-4.0 (m, 1H), 3.96-3.85 (m, 1H), 3.78-3.64 (m, 5H), 3.62-3.59 (m,4H), 3.55 (s, 3H), 3.51-3.22 (m, 8H), 3.15-2.96 (m, 3H), 2.78 (s, 6H),2.18-2.09 (m, 1H), 1.82 (s, 15H), 1.45-1.38 (m, 1H); LCMS calcd forC₂₉H₅₆N₆O₁₆ (M+H⁺) 745.38, found 745.6.

Example 83 Synthesis of the 5″-(2-hydroxy)ethoxy-6′″-MeO₂CHNintermediate (56)

A mixture of Compound 51 (20 mg, 0.0103 mmol) in dry MeOH (3 mL) wastreated with NaBH₃CN (1.0 M in THF, 41.3 μL, 0.0413 mmol). The mixturewas stirred at room temperature overnight until disappearance ofaldehyde. The solvent was removed under reduced pressure and thereaction mixture was diluted with EtOAc (10 mL) and washed with asolution of NaHCO₃ (saturated, 2 mL) and dried over Na₂SO₄. Afterevaporation of the solvents, the residue was purified by silica gelflash column chromatography (48:1 CH₂Cl₂/MeOH) to give the5″-(2-hydroxy)ethoxy derivative as white solid (16 mg, 80%).

[α]_(D)+19.8° (c 0.8, CHCl₃); R_(f) 0.30 (1:1 EtOAc/hex); LCMS calcd forC₁₀₇H₁₀₃N₅O₃₀ (M+H⁺) 1939.1, found 1939.2.

A solution of above 5″-(2-hydroxy)ethoxy derivative (16 mg, 0.0082 mmol)in dry MeOH (2 mL) was treated with a catalytic amount of NaOMe in dryMeOH (1 mL, pH 8-9) and stirred at room temperature for 3 hours tocompletion of reaction. The reaction mixture was neutralized by additionof dry-ice, and the solvent was evaporated to dryness under vacuum. Thecrude product was purified by silica gel flash column chromatography(1:19 MeOH/CH₂Cl₂) to give Compound 56 (8 mg, 72%) as a major product.

[α]_(D)+22.0° (c 0.4, MeOH); R_(f) 0.42 (1:19 MeOH/CH₂Cl₂); LCMS calcdfor C₇₄H₈₈N₆O₂₄ (M+Na⁺) 1364.30, found 1364.5.

Example 84 5″-O-(2-hydroxyethyl)-6′-N-methoxycarbonyl paromomycin (57)

Compound 56 (6 mg, 0.0043 mmol) was dissolved in AcOH—H₂O (4:1, 2 mL)and heated at 60° C. for 2 hours to completion of reaction. The solventwas removed under reduced pressure and the crude product was dissolvedin MeOH—H₂O (1:1, 2 mL), followed by addition of 20% palladium hydroxideon carbon with stirring under an atmosphere of hydrogen (hydrogenballoon). The mixture was filtered through a layer of Celite,concentrated under vacuum, and the residue was dissolved in AcOH—H₂O(2:1, 0.5 mL) and lyophilized to give Compound 57 (4.1 mg, quantitative)as a white solid.

[α]_(D)+36.10° (c 0.20, H₂O); ¹H NMR (400 MHz, D₂O) δ 5.39 (s, 1H), 5.19(s, 1H), 4.90 (s, 1H), 4.30-4.10 (m, 3H), 4.0-3.93 (m, 2H), 3.90-3.82(m, 1H), 3.79-3.55 (m, 12H), 3.51 (s, 3H), 3.40-3.10 (m, 5H), 3.05-2.82(m, 3H), 2.0-1.95 (m, 1H), 1.74 (s, 12H), 1.35-1.25 (m, 1H); LCMS calcdfor C₂₇H₅₁N₅O₁₇ (M+H⁺) 718.33, found 718.5.

Example 85 6′″-N-methoxycarbonyl paromomycin (59)

Following the procedures of the above examples for Compound 57,paromomycin with 6′″-N-methylcarbamate, Compound 59, was prepared forcomparison starting from Compound 58. Compound 58 was obtained followingthe procedure of Example 1 wherein Compound 58 was isolated prior toaddition of benzaldehyde.

¹H NMR (400 MHz, D₂O) δ 5.56 (s, 1H), 5.20 (s, 1H), 5.02 (s, 1H),4.27-4.22 (m, 1H), 4.20-4.16 (m, 1H), 4.13-4.0 (m, 2H), 3.95-3.91 (m,1H), 3.80-3.55 (m, 1H), 3.52 (s, 3H), 3.40-3.36 (m, 3H), 3.25-3.17 (m,2H), 3.15-2.95 (m, 1H), 2.20-2.15 (m, 1H), 1.75 (s, 12H), 1.54-1.43 (m,1H); LCMS calcd for C₂₅H₄₇N₅O₁₆ (M+H⁺) 674.30, found 674.5. ¹³C NMR (125MHz, D₂O) δ 181.7, 159.7, 110.0, 96.5, 96.2, 85.0, 81.9, 80.2, 76.2,73.9, 73.7, 73.3, 73.2, 69.0, 69.5, 68.1, 66.6, 60.7, 60.5, 54.3, 52.9,51.4, 50.4, 49.3, 41.0, 30.6, 23.5.

Example 86 5″-Substituted Partially Protected Paromomycin Analogue (60)

Compound 50 (44 mg, 0.0232 mmol) was dissolved in dry CH₂Cl₂ (3 mL) andcooled at −78° C. Diethylaminosulfur trifluoride (DAST, 3.4 μL, 0.0255mmol) was added dropwise at −78° C., and the reaction mixture wasallowed to come slowly to the room temperature and further stirred for 1hour. The reaction mixture was quenched with a few drops of water at 0°C. and diluted with CH₂Cl₂. The organic layer was washed with water,brine, dried over Na₂SO₄ and concentrated under vacuum. The crudeproduct was purified by silica gel flash column chromatography (2:3EtOAc/hexane) to give the 5″-deoxy fluoro derivative (22 mg, 50%).

[α]_(D)+37.37° (c 0.8, CHCl₃); R_(f) 0.54 (1:1 EtOAc/hexane); ¹⁹F NMR(400 MHz, CDCl₃) Φ 237.4-237.6 (m, F-5); LCMS calcd for C₁₀₅H₉₈FN₅O₂₈(M+H⁺) 1896.64, found 1896.8.

To a solution of the 5″-deoxy fluoro derivative (18 mg, 0.0095 mmol) indry MeOH (2 mL) was treated with a catalytic amount of NaOMe in dry MeOH(1 mL, pH 8-9) and stirred at room temperature for 3 hours to completionof reaction. The reaction mixture was neutralized by addition ofdry-ice, and the solvent was evaporated to dryness under vacuum. Thecrude product which was purified by silica gel flash columnchromatography (1:19 MeOH/CH₂Cl₂) to give Compound 60 (12 mg, 92%).

[α]_(D)+18.0° (c 0.6, MeOH); R_(f) 0.47 (1:19 MeOH/CH₂Cl₂); LCMS calcdfor C₇₀H₇₈FN₅O₂₃ (M+H⁺) 1376.51, found 1377.0.

Example 87 5″-deoxy-5″-fluoro paromomycin (61)

Compound 60 (12 mg, 0.0087 mmol) was dissolved in AcOH—H₂O (4:1, 3 mL)and heated at 60° C. for 2 hours to completion of reaction. The solventwas removed under reduced pressure and the crude material was used inthe next step for hydrogenolysis without further purification.

LCMS calcd for C₆₃H₇₄FN₅O₂₃ (M+H⁺) 1288.48, found 1288.6.

To a solution of above crude material in MeOH—H₂O (1:1, 2 mL) was added20% palladium hydroxide on carbon and the suspension was stirred at roomtemperature overnight under an atmosphere of hydrogen (hydrogenballoon). The mixture was filtered through a layer of Celite,concentrated under vacuum. The residue was dissolved in AcOH—H₂O (2:1,0.5 mL) and lyophilized to give Compound 61 (2.6 mg, 68%) as a whitesolid.

[α]_(D)+33.07° (c 0.26, H₂O); ¹H NMR (400 MHz, D₂O) δ 5.57 (s, 1H), 5.30(s, 1H), 5.17 (s, 1H), 4.54-4.42 (m, 2H), 4.30-4.21 (m, 4H), 4.21-4.17(m, 2H), 4.07-3.91 (m, 2H), 3.82-3.61 (m, 5H), 3.6-3.54 (m, 1H),3.49-3.40 (m, 2H), 3.38-3.16 (m, 4H), 2.38-2.22 (m, 1H), 1.82 (s, 15H),1.70-1.60 (m, 1H); ¹³C NMR (125 MHz, D₂O) δ 182.1, 111.1, 96.8, 96.3,85.6, 81.7, 80.4, 75.2, 74.2, 73.9, 73.6, 73.4, 71.0, 70.3, 69.9, 68.5,68.0, 61.0, 54.7, 51.5, 50.8, 49.7, 41.0, 30.9, 23.9; LCMS calcd forC₂₃H₄₄FN₅O₁₃ (M+H⁺) 618.29, found 618.4.

Example 884′,6′-O-benzylidene-penta-O-tert-butyldimethylsilanyloxy-penta-N-benzyloxycarbonylparomomycin (62)

To a stirred solution of Compound 2 (1.35 g, 0.98 mmol) in drydichloromethane (20 mL) was added 2,4,6-collidine (1.07 g, 8.82 mmol)and TBDMSOTf (1.811 g, 6.86 mmol) at 0° C. Then the reaction mixture wasslowly brought to room temperature and stirred for 12 hours. Few dropsof water were added to quench the excess TBSOTf and the mixture wasextracted with dichloromethane. The organic layer was washed with brine,dried over anhydrous Na₂SO₄ and concentrated. The corresponding crudeproduct was purified by silica gel flash column chromatography to giveCompound 62 (1.048 g, 55%).

[α]_(D)=+16° (c 0.6, CHCl₃). ESI/MS calcd for C₁₀₀H₁₄₉N₅O₂₄Si₅ (M+H⁺)1944.94; found 1946.

Example 89 Synthesis of the Cyclic Arbamate (63)

To a stirred solution of Compound 62 (330 mg, 0.17 mmol) in dry DMF (6mL) was added 60% NaH in mineral oil (8 mg) at 0° C. with stirringcontinued 6 hours at 0° C. A few drops of saturated ammonium chloridesolution were added, followed by extraction with ethyl acetate. Theorganic layer was washed with brine, dried over anhydrous Na₂SO₄ andconcentrated. The corresponding crude product was purified by silica gelflash column chromatography to give Compound 63 (180 mg, 58%) and 120 mg(36%) of starting material, Compound 62.

[α]_(D)=+18° (c 0.5, CHCl₃). ESI/MS calcd for C₉₃H₁₄₁N₅O₂₃Si₅ (M+H⁺)1836.89; found 1837.6

Example 904′,6′-O-benzylidene-penta-O-tert-butyldimethylsilanyloxy-tetra-N-benzyloxycarbonylparomomycin (64)

To a solution of Compound 63 (190 mg, 0.1 mmol) in DMF (7 mL) was added0.7 mL of aqueous LiOH (9 mg, 0.21 mmol) with stirring continued foradditional 3 hours at room temperature. A few drops of saturatedammonium chloride solution was added, followed by extraction with ethylacetate. The organic layer was washed with brine, dried over anhydrousNa₂SO₄ and concentrated. The corresponding crude product was purified bysilica gel flash column chromatography to give Compound 64 (100 mg, 53%)and 50 mg (26%) of starting material, Compound 63.

[α]_(D)=+13° (c 0.3 CHCl₃) ESI/MS calcd for C₉₂H₁₄₃N₅O₂₂Si₅ (M+H⁺)1810.91; found 1811.3.

Example 914′,6′-O-benzylidene-penta-O-tert-butyldimethylsilanyloxy-tetra-N-benzyloxycarbonyl-N-1-habaparomomycin (65)

To a stirred solution of benzyloxy 4-hydroxy aminobutric acid (27 mg,0.11 mmol) and N-hydroxy succinimide (12 mg, 0.11 mmol) in dry THF (2mL) was added DCC (22 mg, 0.11 mmol) with stirring continued for 1 hourat room temperature. To this mixture Compound 64 (95 mg, 0.053 mmol) indry THF (2 mL) and triethyl amine (15 μL, 0.11 mmol) was added andstirred for 12 hours at room temperature. Evaporation of the solventfollowed by purification by silica gel flash column chromatography gaveCompound 65 (80 mg, 74%).

[α]_(D)=+19° (c 0.4, CHCl₃).

Example 92 4′,6′-O-benzylidene-tetra-N-benzyloxycarbonyl-N-1-habaparomomycin (66)

Compound 65 (90 mg, 0.044 mmol) was dissolved in dry pyridine (2 mL),HF.Py (2 mL) was added at 0° C. and the reaction was slowly brought toroom temperature and stirred for 2 days. Water was added and thereaction mixture was extracted with ethyl acetate. The organic layerswere washed with brine and dried over Na₂SO₄ Evaporation of the solventgave the crude material which was purified by column chromatography togive Compound 66 (50 mg, 77%).

[α]_(D)=+20° (c 0.6, CHCl₃). ESI/MS calcd for C₇₄H₈₆N₆O₂₆ (M+H⁺);1475.56; found 1475.7.

Example 93 N-1-haba paromomycin (67)

Compound 66 (29 mg, 0.019 mmol) was stirred in 4 mL of acetic acid/watermixture (4:1) at room temperature for 12 hours and then for anadditional 6 hours at 55° C. To this reaction mixture 20% Pd(OH)₂ (29mg) was added under an atmosphere of hydrogen (balloon) with stirringfor 3 hours. The mixture was filtered over celite and lypholized to givethe Compound 67 (20 mg, 99%).

[α]_(D)=+14.5° (c 0.2, H₂O). ¹H NMR (400 MHz, D₂O) δ 5.84 (s, 1H), 5.44(s, 1H), 5.21 (s, 1H), 5.35 (s, 1H), 4.61 (bs, 1H), 4.49-3.4 (m, 24H),2.26-2.11 (m, 2H), 1.8-1.7 (m, 2H); HRMS calcd for C₂₇H₅₂N₆O₁₆ (M+H⁺):717.34398; found: 717.35175.

Example 94 Synthesis of 2″-O- and 6′-N-Side Chain Paromomycin Analogs

Starting with Compound 68 (the TBDPS protected version of Compound 4prepared according to Examples 2-3 using TBDPS-OTf instead ofTBDMS-OTf), 2″-O- and 6′-N-modified Paromomycin analogs 73(27-O-phenylethylaminoethyl-6′-phenylpropyl neomycin) and 76(N-1-haba-2″-O-phenylethylaminoethyl-6′-phenylpropyl neomycin) wereprepared with and without the 1-HABA group. 3′-4′ dideoxy analogs (onring I) are prepared by similar means starting from Compound 31 inExample 61. The synthetic methods illustrated in this example and incombination with other examples, particularly Examples 1-44, enable thepreparation of a plurality of diverse di and tri-substituted Paromomycinanalogs. Numerous modifications known in the chemical arts are amenableto the synthetic methods disclosed herein to enable even further diverseParomomycin analogs.

Example 95 Synthesis of Paromomycin Analogs Substituted at the 1, 2″ and5″ Positions

Paromomycin analogs substituted at the 1, 2″ and the 5″ positions, suchas N-1-haba-2″-O-phenylethylaminoethyl-5″-fluoro paromomycin (Compound78) and N-1-baba-2″-O-phenylethylaminoethyl-5″-isopropylaminoparomomycin (Compound 80), are prepared following the synthetic methodsillustrated herein and particularly Examples 1-13, 61-77, 78-84 and86-87. Substitution in this pattern without N-1 substitution can beachieved by starting with compounds 8, then removing the TBS group withAcOH, and continuing as shown in this example. 3′-4′ dideoxy analogs (onring I) are prepared by similar means starting from compound 31 inExample 61. Numerous modifications known in the chemical arts, such asfor example variation of chemical functional groups or reactionconditions, are amenable to the synthetic methods disclosed herein. Suchmodifications are intended to be included in the present invention andwill enable even further diverse Paromomycin analogs.

Example 96 Synthesis of Paromomycin Analogs Substituted at the 1, 6′ and5″ Positions

Paromomycin analogs substituted at the 6′ and the 5″ positions with orwithout N-1 substitution, such as N-1-haba-6′-phenylpropyl-5″-fluoroneomycin (Compound 82) and N-1-haba-6′-phenylpropyl-5″-isopropylaminoneomycin (Compound 84), are prepared following the synthetic methodsillustrated herein and particularly Examples 14-44, 78-84 and 86-87.3′-4′ dideoxy analogs (on ring 1) are prepared by similar means startingfrom Compound 31 in Example 61. Numerous modifications known in thechemical arts, such as for example variation of chemical functionalgroups or reaction conditions, are amenable to the synthetic methodsdisclosed herein. Such modifications are intended to be included in thepresent invention and will enable even further diverse Paromomycinanalogs.

Example 97 Synthesis ofN-1-haba-2″-O-phenylethylaminoethyl-3′,4′-dideoxy neomycin (87)

To a stirred solution of 38 (100 mg, 0.067 mmol) and CbzOSu (33 mg,0.133 mmol) in dioxane (10 mL) was added aqueous saturated NaHCO₃ (5 mL)and continued to stir for 6 h. Saturated ammonium chloride solution wasadded, followed by extraction with ethyl acetate. The organic layer waswashed with saturated brine and dried over anhydrous Na₂SO₄, followed byconcentration of the solvent yielded the corresponding crude product.The crude material was purified by flash column chromatography to yieldpure 85 (61 mg, 56%). ESUMS calcd for C₈₅H₁₀₁N₇O₂₆ (M+H⁺): 1636.74;found: 1636.7.

To a stirred solution of 85 (60 mg, 0.037 mmol) in pyridine (5 mL) wasadded TsCl (9 mg, 0.046) and continued to stir overnight. Few drops ofwater were added, followed by extraction with ethyl acetate. The organiclayer was washed with saturated CuSO₄ solution, brine and dried overanhydrous Na₂SO₄, followed by concentration of the solvent yielded thecorresponding crude product. This crude material in dry DMF was addedNaN₃ (24 mg, 0.37 mmol) and heated at 70° C. for 12 h. Few drops ofsaturated ammonium chloride were added, followed by extraction withethyl acetate. The organic layer was washed with water, brine and driedover anhydrous Na₂SO₄, followed by concentration of the solvent yieldedthe corresponding crude product. This material was purified by flashcolumn chromatography to yield the pure 86 (21 mg, 34%). In addition tothe product, some regioisomeric product (15 mg, 24%) and starting freehydroxyl compound (10 mg) were isolated. ESI/MS calcd for C₈₅H₁₀₀N₁₀O₂₅(M+H⁺): 1661.76; found: 1661.9.

To a stirred solution of 86 (20 mg, 0.012 mmol) in 2 mL of aceticacid/water mixture (4:1) and 0.5 mL of methanol was added 20% Pd(OH)₂(20 mg) at room temperature and stirred for 6 h under hydrogenatmosphere (balloon). Then filtered over celite and lypholized to give87 as acetate salt (14 mg, 93%). [α]_(D)=+27.1° (c 0.2, H₂O). ¹H NMR(400 MHz, D₂O) δ 7.4-7.1 (m, 5H), 5.5 (s, 1H), 5.28 (s, 1H), 5.08 (s,1H), 4.5-4.4 (m, 1H), 4.2-4.0 (m, 5H), 3.9-3.6 (m, 9H), 3.5-3.1 (m,11H), 3.0-2.8 (m, 5H), 2.1-1.4 (m, 8H); ESI/MS calcd for C₃₇H₆₆N₈O₁₃(M+H⁺): 830.47493; found: 830.48221.

Example 98 Synthesis of Paromomycin Analogs Substituted at the 1 and 5″Positions

Paromomycin analogs substituted at the 1′ and the 5″ positions areprepared following the synthetic methods illustrated herein andparticularly Examples 45-59, 61-77, 78-84, 86-87 and 89-93. 3′-4′dideoxy analogs (on ring I) are prepared by similar means starting fromCompound 31 in Example 61. Numerous modifications known in the chemicalarts, such as for example variation of chemical functional groups orreaction conditions, are amenable to the synthetic methods disclosedherein. Such modifications are intended to be included in the presentinvention and will enable even further diverse Paromomycin analogs.

Example 99 Synthesis of Paromomycin Analogs Substituted at the 6 and 2″Positions

Paromomycin analogs substituted at the 6′ and the 6 positions areprepared following the synthetic methods illustrated herein andparticularly Examples 1-13, 53-59 and 61-77. 3′-4′ dideoxy analogs (onring I) are prepared by similar means starting from Compound 31 inExample 61. Numerous modifications known in the chemical arts, such asfor example variation of chemical functional groups or reactionconditions, are amenable to the synthetic methods disclosed herein. Suchmodifications are intended to be included in the present invention andwill enable even further diverse Paromomycin analogs.

Example 100 Synthesis of Paromomycin Analogs Substituted at the 6 and 5″Positions

Paromomycin analogs substituted at the 6 and the 5″ positions areprepared following the synthetic methods illustrated herein andparticularly Examples 53-59, 78-84 and 86-87. 3′-4′ dideoxy analogs (onring I) are prepared by similar means starting from Compound 31 inExample 61. Numerous modifications known in the chemical arts, such asfor example variation of chemical functional groups or reactionconditions, are amenable to the synthetic methods disclosed herein. Suchmodifications are intended to be included in the present invention andwill enable even further diverse Paromomycin analogs.

Example 101 Synthesis of Paromomycin Analogs Substituted at the 6′ and 6Positions

Paromomycin analogs substituted at the 6′ and the 6 positions areprepared following the synthetic methods illustrated herein andparticularly Examples 14-44 and 53-59. 3′-4′ dideoxy analogs (on ring I)are prepared by similar means starting from Compound 31 in Example 61.Numerous modifications known in the chemical arts, such as for examplevariation of chemical functional groups or reaction conditions, areamenable to the synthetic methods disclosed herein. Such modificationsare intended to be included in the present invention and will enableeven further diverse Paromomycin analogs.

Example 102 Coupled Bacterial Transcription/Translation Assay

The DNA template, pBestLuc™ (Promega), is a plasmid containing areporter gene for firefly luciferase fused to a strong tac promoter andribosome binding site. Messenger RNA from 1 μg pBestLuc is transcribedand translated in E. coli S30 bacterial extract in the presence orabsence of test compound. Compounds are tested in a black 96 wellmicrotiter plate with an assay volume of 35 μL. Each test well contains:5 μL test compound, 13 μL S30 premix (Promega), 4 μL 10× complete aminoacid mix (1 mM each), 5 μL E. coli S30 extract and 8 μL of 0.125 μg/μLpBestLuc™. The transcription/translation reaction is incubated for 35minutes at 37° C. followed by detection of functional luciferase withthe addition of 30 μL LucLite™ (Packard). Light output is quantitated ona Packard TopCount.

Example 103 Mass Spectrometry Based Binding Asay

Screening was performed by measuring the formation of non-covalentcomplexes between a single ligand or ligand mixture and the appropriateRNA target, such as for example the 16S Kd and 18S Kd ribosomalsubunits, along with suitable control structured RNA target(s)simultaneously using a 9.4 T FT-ICR mass spectrometer as detector. Fullexperimental details of the assay for have been described in relatedliterature (Sannes-Lowery, et al. in TrAC, Trends Anal. Chem. 2000, 19,481-491; Sannes-Lowery, et al. in Anal. Biochem. 2000, 280, 264-271; andGriffey, R. H.; Sannes-Lowery, K. A.; Drader, J. J.; Mohan, V.; Swayze,E. E. et al. Characterization of Low Affinity Complexes Between RNA andSmall Molecules Using Electrospray Ionization Mass Spectrometry. J. Am.Chem. Soc. 2000, 122, 9933-9938).

In a typical experiment, 10 μL of an aqueous solution containing 100 mMammonium acetate buffer, 2.5 or 5 μM of each RNA, and 33% isopropylalcohol (to aid ion desolvation) was prepared with differentconcentrations of each ligand or ligand mixture Samples were introducedinto the electrospray ionization source (negative ionization mode) at 1μL/min and ions were stored for 1 sec in an RF-only hexapole followingdesolvation. The abundances were integrated from the respective ions forfree RNA and the ligand-RNA complex. The primary (1:1 RNA:ligand) andsecondary (1:2 complex, if observed). KD values were detecniined bytitrating a single ligand through a concentration range of 0.25-25 μMwith an RNA target concentration of 0.10 μM. The peak ratios weremeasured at each concentration, then a plot of complex/free RNA versusconcentration of ligand added was fitted to a second (or higher) orderbinding polynomial to determine the KD.

Example 104 In Vitro Antibacterial Activity Determination of MinimumInhibitory Concentrations (MICs)

The MIC assays are carried out in 150 μL volume in duplicate in 96-wellclear flat-bottom plates. The bacterial suspension from an overnightculture growth in appropriate medium is added to a solution of testcompound in 4% DMSO in water. Final bacterial inoculum is approximately10⁵-10⁶ CFU/well. The percent growth of the bacteria in test wellsrelative to that observed for a well containing no compound is deteimined by measuring absorbance at 595 nm (A₅₉₅) after 24 h. The MIC isdetermined as a range of single compound where the complete inhibitionof growth is observed at the higher concentration and cells are viableat the lower concentrations. Both ampicillin and tetracycline are usedas antibiotic-positive controls in each screening assay for S. pyogenes,E. coli imp-, E. coli, S. aureus, E. faecalis, K. pneumoniae and P.vulgaris. Ciprofloxacin is used as an antibiotic positive control ineach screening assay for P. aeruginosa.

Example 105 Representative Aminoglycoside Compounds

The following compounds were prepared using methods illustrated in theprevious examples. The compounds were analyzed for their activity usingFTICR mass spectrometry (for 16S Kd, run at 100 nM RNA, except thosemarked with an asterisk were run at 500 nM RNA) and a bacterialtranscription/translation assay, such as described herein. The compoundswere also examined in standard bacterial assays against E. Coli and S.Aureus to determine activities. Data marked with “^(b)”⁻⁻ initiallytested MIC <1.5 uM, but retested higher.

18S Kd 16S Kd Trans/Trans MIC (uM) Compound# (uM) (uM) IC50 (uM) E.Coli. S. Aureus  9h NA 9.2 1.0 >50 25-50  9i NA 1.3 0.2 25-50 2-3  9g NA0.9* 0.2 12-52  6-12  9c NA 0.4* 0.3 25-50 3-6  9f NA 0.3 0.4 12-25 2-3 9d NA 0.7 0.3  6-12 2-3 61 NA 1.1 0.3 >50 12-25 13 NA 2.7 0.3 25-50 3-614a NA 3.8 0.3 >50  6-12 30a 58 19 0.2  6-12 12-25 30b 18 9.2 0.1  6-12 6-12 12a 7.3 0.9 0.1 12-25  6-12  9j 5.0 0.1 0.1 1.5-3  3-6  9k 2.6 0.30.04 1.5-3  3-6 54 68 4.1 0.3  6-12 25-50  9e 11 3.9 0.2 >100  >100  9a3.5 0.6 0.1  3-6^(b) 0.6-1   9b 9.4 0.9 0.1   6-12^(b) 0.6-1   9m 0.90.1 0.1 12-25 1-2  9n 0.4 0.1 0.2 3-6 3-5  9l NA NA 0.1  50-100  6-12 9t 0.3 0.02 0.7  6-12 0.6-1   9r 1.8 0.6 0.3 12-25 2-3  9o 1.0 0.2 0.2 6-12 0.6-1   9p 1.3 0.1 1.1 3-6 0.3-0.6  9q 0.3 0.1 0.8 3-6 3-5  9s 0.50.1 0.2 3-6 0.3-0.6  9u 22 5 0.4 12-25 1-2  9x 6.3 1.0 0.1 3-5 0.6-1.2 9z 6.4 0.7 0.1 3-5 0.6-1.2 14c 59 40 0.2 10-20 3-5  9ak 3.5 0.7 0.310-20  1-3.

Example 106 Representative Aminoglycoside Compounds

The following compounds were prepared using methods illustrated in theprevious examples. The compounds were analyzed for their activity usingFTICR mass spectrometry and a bacterial transcription/translation assay,such as described herein. The compounds were also examined in standardbacterial assays against E. Coli and S. Aureus to determine activities.

18S Kd 16S Kd Trans/Trans MIC (uM) Compound# (uM) (uM) IC50 (uM) E.Coli. S. Aureus paromomycin 4.8 0.6 0.1 20-40 3-5  9y NA NA 0.2 10-20 5-10  9aa 3.5 0.9 1.5 20-40  5-10  9v 5.8 2.5 0.3 10-20 0.6-1   9w 1.73.2 0.4 20-40 1-3 11 6.1 1.5 0.4 10-20 1-3 10 2.5 3.9 0.4 10-20 3-5

Neomycin B was also tested in the assays described. The 16S and 18S Kdwere 0.04 and 0.3 μM, respectively. The Trans/Trans IC50 was 0.2 μM, andthe MIC for E. Coli and S. Aureus were 1.3-2.5 and 0.6-1.3,respectively.

Example 107 Representative Aminoglycoside Compounds

The following compounds were prepared using methods illustrated in theprevious examples. The compounds were examined in standard bacterialassays against E. Coli and S. Aureus to determine activities. Ifpresent, “N.D.” indicates “no data”.

MIC (uM) Compound# E. Coli. S. Aureus  9ac >10 1.3-2.5  9ab >10  5-10 9ad >10 2.5-5   9ae N.D. N.D.  9af >10 2.5-5   9ah  5-10 0.6-1.2 67 5-10 1.3-2.5 12b 20-40  5-10 14b 20-40 3-5 14c 10-20 3-5 30c >100 12-25

Example 108 Representative Aminoglycoside Compounds

The following compounds were prepared using methods illustrated in theprevious examples. The compounds were analyzed for their activity usingFTICR mass spectrometry and a bacterial transcription/translation assay,such as described herein. The compounds were also examined in standardbacterial assays against E. Coli and S. Aureus to determine activities.

18S Kd 16S Kd Trans/Trans MIC (uM) Compound# (uM) (uM) IC50 (uM) E.Coli. S. Aureus 15d 1.4 0.01 0.3  6-12 12-25 15a 0.7 0.6 0.3 3-6 2-3 15e1.2 0.4 0.2 2-3 2-3 15i 1.7 0.6 0.2 12-25 3-6 15h 1.2 1.2 0.2 2.5-5 1.3-2.5 15b 1.4 1.6 0.2 2.5-5  1.3-2.5 15f 6.0 6.3 0.0 2.5-5  10-20 15j2.2 0.5 0.2 1.3-2.5 0.3-0.6 15c 3.0 3.6 0.3 20-40 10-20

Example 109 Representative Aminoglycoside Compounds

The following compounds were prepared using methods illustrated in theprevious examples. The compounds were examined in standard bacterialassays against E. Coli and S. Aureus to determine activities. Ifpresent, “N.D.” indicates “no data”.

MIC (uM) Compound# E. Coli. S. Aureus 15g 2.5-5  0.6-1.2 15k 2.5-5 0.3-0.6 15l 0.6-1.2 0.3-0.6 15r 1.3-2.5 0.3-0.6 15v 1.3-2.5 0.3-0.6 15n1.3-2.5 0.3-0.6 15p 1.3-2.5 0.2-0.3 15t  5-10 0.6-1.2 15m 1.3-2.50.3-0.6 15s 1.3-2.5 0.3-0.6 15w 1.3-2.5 1.3-2.5 15x 1.3-2.5 0.3-0.6 15q1.3-2.5 0.3-0.6 15n 0.6-1.2 0.2-0.3 15o 0.6-1.2 0.2-0.3 15z 1.3-2.50.3-0.6 15aa 1.3-2.5 0.3-0.6 15y 1.3-2.5 0.3-0.6

Example 110 Representative Aminoglycoside Compounds

The following compounds were prepared using methods illustrated in theprevious examples. The compounds were also examined in standardbacterial assays against E. Coli, S. Aureus, P. aurginosa, K.pneumoniae, P. vulgaris, and A. baumannii to deteiniine activities. Eachof the bacterial cultures that are available from ATCC (www.atcc.org) isidentified by its ATCC number. A. baumannii is gentamicin sensitiveAcinetobacter baumannii #2 from Walter Reed.

MIC (uM) E. coli S. aureus P. aurginosa P. aurginosa K. pneumoniae P.vulgaris A. baumannii ATCC ATCC ATCC ATCC ATCC ATCC WReed Compound#25922 13709 25416 29248 10031 8427 2 9p - sample 1 5-10 10-20 >40 >401.3-2.5 2.5-5  2.5-5  9p - sample 2 >40 1.2-2.5 >40 >40 >40 20-40 >4015j 1.3-2.5  10-20 >40 >40   <0.6 1.3-2.5 1.3-2.5 9ae >40 10-20 5-10 >40  5-10 20-40 20-40 67 2.5-5   20-40 1.3-2.5  5-10 0.6-1.3  5-102.5-5  46a 0.6-1.3  0.6-1.2 0.6-1.3 1.3-2.5   <0.6 0.6-1.3 0.6-1.3 46b5-10 1.2-2.5 1.3-2.5  5-10 1.3-2.5  5-10 2.5-5  9ag 10-20  0.6-1.210-20 >40 2.5-5   10-20 10-20 9aj 5-10 <0.6 >40 >40 1.3-2.5  5-10 2.5-5 9ai 5-10 1.2-2.5 2.5-5  20-40 2.5-5    5-10 10-20

Example 111 Representative Aminoglycoside Compounds

The following compounds were prepared using methods illustrated in theprevious examples. The compounds were also examined in standardbacterial assays against E. Coli, S. Aureus, P. aurginosa, K.pneumoniae, P. vulgaris, and A. baumannii to determine activities. Eachof the bacterial cultures that are available from ATCC (www.atcc.org) isidentified by its ATCC number. A. baumannii is gentamicin sensitiveAcinetobacter baumannii #2 from Walter Reed.

MIC (uM) E. coli S. aureus P. aurginosa P. aurginosa K. pneumoniae P.vulgaris A. baumannii ATCC ATCC ATCC ATCC ATCC ATCC WReed Compound#25922 13709 25416 29248 10031 8427 2 Paromomycin  5-10 10-20 >40 >400.6-1.3 >40 2.5-5  15j 1.3-2.5 0.3-0.6 >40 >40 0.6-1.3 >40 20-40 15l1.3-2.5 0.2-0.3 >40 >40 0.6-1.3 >40 10-20 15u 1.3-2.5 0.3-0.6 >40 >400.6-1.3 >40 10-20 15o 1.3-2.5 0.3-0.6 >40 >40 0.6-1.3 >40 10-20 9p -sample 1  5-10 0.6-1.3 >40 >40 2.5-5  >40 >40 67  5-10 1.3-2.5 10-205-10 0.6-1.3 2.5-5  5-10 46a 1.3-2.5 0.2-0.3 10-20 5-10 0.3-0.6   5-1010-20 39 2.5-5  0.6-1.3 >40 >40 1.3-2.5 >40 20-40

Example 112 Representative Aminoglycoside Compounds

The following compound was prepared using methods illustrated in theprevious examples. The compound was also examined in standard bacterialassays against E. Coli, S. Aureus, P. aurginosa, K. pneumoniae and A.baumannii to determine activities. Each of the bacterial cultures thatare available from ATCC (www.atcc.org) is identified by its ATCC number.

MIC (uM) S. P. K. A. E. coli aureus aurginosa pneumoniae baumannii ATCCATCC ATCC ATCC ATCC Compound# 25922 29213 27853 10031 19606 87 <16 <16<16 <16 <16

Example 113

Staphylococcus aureus (Smith Strain ATCC 13709) Mouse Protection Assay

Two of the novel aminoglycoside compounds of the invention were examinedfor their anitbacterial activity against staphylococcus aureus. Micewere fed with autoclaved commercial food pellets and sterile water adlibitum. Animals were inoculated intraperitoneally with 0.5 mL/mouse ofthe indicated concentration of S. aureus (ATCC 13709) containing 10%mucin. There were 10 mice in each treatment group and compounds wereadministered subcutaneously one and 3 hour after infection. Compounds15a (R₄═R₅═CH₃) and 15j (R₄═H, R₅═(CH₂)₂C₆H₅)) were used at 75 mg/kg,37.5 mg/kg, 18.8 mg/kg, 9.4 mg/kg, 4.7 mg/kg, 2.3 mg/kg, 1.17 mg/kg and0.5 mg/kg. Amakacin, paromomycin and neomycin were used as the positivecontrols at concentrations of 2 mg/kg, 1 mg/kg and 0.5 mg/kg.

Staph Conc. Antibiotic conc. # Dead mice/Total mice in group 10⁹ 0 5/5(10% Mucin) 10⁸ 0 5/5 (10% Mucin) 10⁷ 0 1/5 (10% Mucin) 10⁶ 0 0/5 (10%Mucin)  0 0 0/10 (10% Mucin) 10⁶ 0 9/10 (10% Mucin) 10⁷ 0 9/10 (10%Mucin) 10⁶ Amikacin 2 mg/kg 8/10 (10% Mucin) 10⁶ Amikacin 1 mg/kg 10/10(10% Mucin) 10⁶ Amikacin 0.5 mg/kg 8/10 (10% Mucin) 10⁶ Paromomycin 2mg/kg 9/10 (10% Mucin) 10⁶ Paromomycin 1 mg/kg 10/10 (10% Mucin) 10⁶Paromomycin 0.5 mg/kg 10/10 (10% Mucin) 10⁶ Neomycin 2 mg/kg 4/10 (10%Mucin) 10⁶ Neomycin 1 mg/kg 10/10 (10% Mucin) 10⁶ Neomycin 0.5 mg/kg7/10 (10% Mucin) 10⁶ 15a 75 mg/kg 0/10 (10% Mucin) 10⁶ 15a 37 mg/kg 0/10(10% Mucin) 10⁶ 15a 18 mg/kg 0/10 (10% Mucin) 10⁶ 15a 9 mg/kg 0/10 (10%Mucin) 10⁶ 15a 4.5 mg/kg 1/10 (10% Mucin) 10⁶ 15a 2 mg/kg 7/10 (10%Mucin) 10⁶ 15a 1 mg/kg 7/10 (10% Mucin) 10⁶ 15a 0.5 mg/kg 8/10 (10%Mucin) 10⁶ 15j 75 mg/kg 0/10 (10% Mucin) 10⁶ 15j 37 mg/kg 0/10 (10%Mucin) 10⁶ 15j 18 mg/kg 0/10 (10% Mucin) 10⁶ 15j 9 mg/kg 0/10 (10%Mucin) 10⁶ 15j 4.5 mg/kg 0/10 (10% Mucin) 10⁶ 15j 2 mg/kg 0/10 (10%Mucin) 10⁶ 15j 1 mg/kg 0/10 (10% Mucin) 10⁶ 15j 0.5 mg/kg 7/10 (10%Mucin)

In a similar experiment, compounds 9p (R₆═CH₂C₆H₅) and 9b (R₆=3-pyridyl)were used at 75 mg/kg, 37 mg/kg, 18 mg/kg, 9 mg/kg, 4.5 mg/kg, 2 mg/kg,1 mg/kg, 0.5 mg/kg, 0.25 mg/kg, and 0.1 mg/kg in the staphylococcusaureus protection assay. Test compound was administered as an aqueousbuffer solution (phosphate buffered saline (PBS), pH=7.4). The data inthe table below clearly indicate that both 9p and 9b are effective atpreventing lethal bacterial infections in mice, with 9p being protectiveat doses as small as 0.25 mg/kg.

Staph Conc. Antibiotic conc. # Dead mice/Total mice in group 10⁶ 9p 75mg/kg 0/10 (10% Mucin) 10⁶ 9p 37 mg/kg 0/10 (10% Mucin) 10⁶ 9p 18 mg/kg0/10 (10% Mucin) 10⁶ 9p 9 mg/kg 0/10 (10% Mucin) 10⁶ 9p 4.5 mg/kg 0/10(10% Mucin) 10⁶ 9p 2 mg/kg 0/10 (10% Mucin) 10⁶ 9p 1 mg/kg 0/10 (10%Mucin) 10⁶ 9p 0.5 mg/kg 0/10 (10% Mucin) 10⁶ 9p 0.25 mg/kg 1/10 (10%Mucin) 10⁶ 9p 0.1 mg/kg 5/10 (10% Mucin) 10⁶ 9b 75 mg/kg 0/10 (10%Mucin) 10⁶ 9b 37 mg/kg 0/10 (10% Mucin) 10⁶ 9b 18 mg/kg 0/10 (10% Mucin)10⁶ 9b 9 mg/kg 0/10 (10% Mucin) 10⁶ 9b 4.5 mg/kg 0/10 (10% Mucin) 10⁶ 9b2 mg/kg 0/10 (10% Mucin) 10⁶ 9b 1 mg/kg 0/10 (10% Mucin) 10⁶ 9b 0.5mg/kg 3/10 (10% Mucin) 10⁶ 9b 0.25 mg/kg 6/10 (10% Mucin) 10⁶ 9b 0.1mg/kg 7/10 (10% Mucin)

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification areincorporated herein by reference, in their entirety to the extent notinconsistent with the present description.

1-36. (canceled)
 37. A pharmaceutical composition comprising apharmaceutically acceptable carrier or excipient and a compound havingthe following formula VI:

or a prodrug or pharmaceutically acceptable salt thereof, wherein: Q₁ is—NR₁₀R₁₁; Q₂ is

each Q₃ and Q₄ is —OR₇; Q₅ is —OR₈; each of R₁, R₆, R₇ and R₈ is, ineach instance, H; each R₂ and R₁₀ is, independently, H, C₁-C₁₂ alkyl orsubstituted C₁-C₁₂ alkyl; R₁₁ is cyano, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl or —(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁; L₁ isS, O or NJ₁; n is an integer from 1 to 8; m is 0 or 1; nn is 0 or aninteger from 1 to 8; mm is 1 or 2; E₁ is H, hydroxyl, halogen, cyano,—NJ₁J₂, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl,heteroaryl, substituted heteroaryl, a heterocyclic radical, asubstituted heterocyclic radical or a substituted or unsubstituted monoor poly cyclic structure that is unsaturated, partially saturated orfully saturated and optionally includes one or more heteroatoms selectedfrom O, N and S; each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl,substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl,C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substitutedC₅-C₂₀ aryl, —C(═O)—X, a heterocyclic radical or a substitutedheterocyclic radical; each X is, independently, H, C₁-C₁₂ alkyl orsubstituted C₁-C₁₂ alkyl; and Z₁ and Z₂ are each, independently, H or—OH.
 38. A pharmaceutical composition according to claim 37, wherein mmis
 1. 39. A pharmaceutical composition according to claim 37, wherein mmis
 2. 40. A pharmaceutical composition according to claim 37, whereineach R₂ is H.
 41. A pharmaceutical composition according to claim 37,wherein Z₁ is —OH.
 42. A pharmaceutical composition according to claim37, wherein R₁₀ is H.
 43. A pharmaceutical composition according toclaim 42, wherein R₁₁ is substituted C₁-C₁₂ alkyl.
 44. A pharmaceuticalcomposition according to claim 38, wherein: R₁₀ is H; R₁₁ is substitutedC₁-C₁₂ alkyl; each R₂ is H; and Z₁ is —OH.
 45. A pharmaceuticalcomposition according to claim 45, wherein mm is
 1. 46. A pharmaceuticalcomposition according to claim 45, wherein mm is
 2. 47. A pharmaceuticalcomposition according to claim 37, wherein said compound of formula VIhas the configuration:


48. A pharmaceutical composition according to claim 47, wherein mm is 1.49. A pharmaceutical composition according to claim 47, wherein mm is 2.50. A pharmaceutical composition according to claim 47, wherein each R₂is H.
 51. A pharmaceutical composition according to claim 47, wherein Z₁is —OH.
 52. A pharmaceutical composition according to claim 47, whereinR₁₀ is H.
 53. A pharmaceutical composition according to claim 52,wherein R₁₁ is substituted C₁-C₁₂ alkyl.
 54. A pharmaceuticalcomposition according to claim 47, wherein: R₁₀ is H; R₁₁ is substitutedC₁-C₁₂ alkyl; each R₂ is H; and Z₁ is —OH.
 55. A pharmaceuticalcomposition according to claim 54, wherein mm is
 1. 56. A pharmaceuticalcomposition according to claim 54, wherein mm is
 2. 57. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier orexcipient and a compound having the following formula I:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof,wherein: Q₁ is azido or —NR₁₀R₁₁; Q₂ is —NR₁₀R₁₂; each Q₃ and Q₄ is—OR₇; Q₅ is H, halogen, cyano, azido, —OR₈, —NR₂R₃, a protected aminogroup or a nitrogen containing heterocyclic radical which optionallyincludes one or more additional heteroatoms selected from N, O and Swherein the heterocyclic radical is covalently linked through saidnitrogen atom; each R₁ is, independently, H or a hydroxyl protectinggroup; each R₂ is, independently, H, an amino protecting group, C₁-C₁₂alkyl or substituted C₁-C₁₂ alkyl; each R₃ is, independently, H, anamino protecting group, cyano, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl,C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substitutedC₂-C₁₂ alkynyl or —(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁; each R₆ is,independently, H or an amino protecting group; each R₇ is,independently, H, a hydroxyl protecting group or—(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁; R₈ is H, a hydroxyl protecting group,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl or—(CH₂)_(n)-(L₁)_(m)-(CH₂)_(nn)-E₁; each R₁₀ is, independently, H, C₁-C₁₂alkyl or substituted C₁-C₁₂ alkyl; each R₁₁ is, independently, cyano,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl or—(CH₂)_(n)-(L₄)_(m)-(CH₂)_(nn)-E₁; and R₁₂ is a group having thefollowing formula III:

each L₁ is, independently, S, O or NJ₁; L₂ is CH or N; each n is,independently, an integer from 1 to 8; each in is, independently, 0 or1; each nn is, independently, 0 or an integer from 1 to 8; mm is 1 or 2;each E₁ is, independently, H, hydroxyl, halogen, cyano, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl,C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heteroaryl, substitutedheteroaryl, a heterocyclic radical, a substituted heterocyclic radicalor a substituted or unsubstituted mono or poly cyclic structure that isunsaturated, partially saturated or fully saturated and optionallyincludes one or more heteroatoms selected from O, N and S; each J₁ andJ₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, —C(═O)—X, a heterocyclicradical or a substituted heterocyclic radical; each X is, independently,H, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl; each Z₁ and Z₂ is,independently, H, hydroxyl or a protected hydroxyl; and Z₃ is a grouphaving the following formula

and wherein when Q₂ is —N(H)C(═O)C(H)(OH)CH₂CH₂NH₂ then Q₁ is other than—N(H)CH₃ or —N(H)CH₂CH₃ and Q₅ is other than —N(H)C(═NH)NH₂ or—N(H)CH═NH₂.
 58. A pharmaceutical composition according to claim 57,wherein one of Z₁ and Z₂ is H.
 59. A pharmaceutical compositionaccording to claim 57, wherein: each of said substituted groups, is,independently, mono or poly substituted with optionally protectedsubstituent groups independently selected from C₁-C₁₂ alkyl, substitutedC₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl or substituted C₅-C₂₀aryl, heterocyclic radical, substituted heterocyclic radical,heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical, substitutedC₅-C₇ alicyclic radical, halogen, —OJ₃, —NJ₁J₂, —SJ₃, —N₃, —COOH,—C(═O)—X, —CN, —S(═O)₂—X, —S(═O)—X, —C(═O)—NJ₁J₂, —N(H)C(═O)-J₁,—N(J₁)—(CH₂)_(nm)—OJ₃ and —N(J₁)—(CH₂)_(nm)—NJ₁J₂ and a substituted orunsubstituted mono or poly cyclic structure that is unsaturated,partially saturated or fully saturated and optionally includes one ormore heteroatoms selected from O, N and S; each J₃ is, independently, H,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂aminoalkyl, substituted C₁-C₁₂ aminoalkyl or a hydroxyl protectinggroup; and nm is an integer from 1 to
 20. 60. A pharmaceuticalcomposition according to claim 57, wherein said compound of formula Ihas the configuration:


61. A pharmaceutical composition according to claim 57, wherein saidcompound of formula I has the configuration: